Multi-domain enzymes having cutinase activity, compositions comprising same and uses thereof

ABSTRACT

An isolated chimeric recombinant protein has cutinase activity. The protein includes a cutinase catalytic domain and a polymer binding domain operably linked by a proline/threonine-rich linker domain. The proline/threonine-rich linker domain includes at least 50% proline or threonine residues over a stretch of 15 to 55 consecutive amino acids.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.15/044,715, filed Feb. 16, 2016, which claims the benefit of U.S.Provisional Application No. U.S. 62/116,761 filed Feb. 16, 2015. Thecomplete disclosures of these prior applications are incorporated byreference in their entirety.

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference the Sequence Listingsubmitted as an ASCII text filed via EFS-Web. The Sequence Listing isprovided as a file entitled Sequence Listing_RBCA088.001C1.txt, createdon Jul. 28, 2017, which is 91 Kb in size.

FIELD

The present invention relates to enzymes with cutinase activity andmethods for transgenic expression thereof, and various uses thereof.More specifically, the present invention is concerned with enzymes withcutinase activity having a modular organization, methods relating totheir transgenic expression, compositions comprising same, and usesthereof.

BACKGROUND

Cutinases (EC 3.1.1.74) are hydrolytic enzymes, i.e. hydrolases, thatdegrade cutin, a component of the plant cuticle that, in addition towaxes, constitute the outermost continuous membrane or “skin” of theprimary parts of higher plants. Cutin is a polyester, largely composedof saturated 016 (palmitic) acids and unsaturated 018 fatty acidscross-linked by ester bonds; the actual composition of which depends onthe plant species. The cuticle primarily protects plants from water lossor other environmental stresses, thereby acting as a formidable barrier.

Cutinases have been found predominantly in phytopathogenic fungi assecreted enzymes to facilitate their entry into the host plant. Theprototypical cutinase is the FsCUT derived from Fusarium solani f. sp.pisi (aka Nectria haematococca) that infects peas. This enzyme plays arole in plant pathogenesis as a virulence factor, but not exclusivelysince hydrophobic surface binding proteins, called hydrophobins, arealso involved. FsCUT is a 230-amino acid enzyme synthesized with a 31amino-acid signal peptide, with the mature, secreted portion having amolecular mass of 21,600 Da (Soliday et al., 1984). Its structurebelongs to the α/β class of hydrolases containing the classicalSer-His-Asp triad (S120, H188 and D175) for catalysis (Martinez et al.,1992).

Owing to the hydrolytic, esterification or transesterificationactivities, both naturally-occurring or genetically-modified cutinasesof Fusarium and other organisms, have been produced and applied tovarying extent in industries such as food, laundry and detergent,textiles, recycling, and polymer manufacturing.

SUMMARY

The present description stems from the surprising discovery that membersof a family of multi-domain enzymes having cutinase activity may haveadvantageous properties for various industrial applications. In someimplementations, these advantageous properties may include improvedstability in the presence of an oxidizing agent, improved stability inthe presence of an organic solvent, and/or improved enzymatic activityat a broader range of temperatures, relative to prototypicalsingle-domain cutinases.

This family of multi-domain enzymes having cutinase activity includes anendogenous enzyme (KrCUT) from the actinobacterium Kineococcusradiotolerans, a highly radiation-resistant actinobacterium isolatedfrom nuclear waste at the Savannah River Site in Aiken, S.C., as well asenzymes from Cellulomonas bogoriensis, Cellulomonas cellasea, andCellulomonas flavigena. This family of enzymes differs from prototypicalcutinases such as those from the filamentous fungus Fusarium solani, inthat they have a multi-domain modular organization in the form of acutinase catalytic domain that has an extra C-terminal segmentcontaining a threnonine/proline-rich (T/P-rich) linker domain, andpolymer binding domain. Recombinant proteins having cutinase activityhaving different improved properties and substrate preferences can thusbe engineered using cutinase catalytic domains, linker domains, andpolymer binding domains present in cutinases from different organisms.

In some implementations, the present description relates to one or moreof the following items.

1. An isolated recombinant protein having cutinase activity, saidprotein comprising: (a) a cutinase catalytic domain; (b) aproline/threonine-rich linker domain positioned C-terminal of domain(a); and (c) a polymer binding domain positioned C-terminal of domain(b).2. The isolated recombinant protein of item 1, wherein said protein, ascompared to a corresponding native protein comprising domain (a):

-   -   (i) has improved stability to a bleaching agent which is:        -   (1) an oxidizing agent which is at least one of: hydrogen            peroxide, a peroxide other than hydrogen peroxide, a            non-peroxide oxidizing agent, ozone, sodium percarbonate,            sodium perborate, or any combination thereof; and/or        -   (2) a reducing agent which is at least one of: sodium            dithionite, sodium dithionite, sulfur dioxide, a sulfite, a            bisulfite, a sodium borohydride, or any combination thereof;    -   (ii) has improved stability to an organic solvent which is at        least one of: methanol, 2-propanol, or another alcohol, dimethyl        sulfoxide (DMSO), dimethylformamide (DMF), acetone, or another        organic solvent, or any combination thereof;    -   (iii) has a broader temperature activity range; or    -   (iv) any combination of (i) to (iii).        3. The isolated recombinant protein of item 1 or 2 which is a        chimeric recombinant protein, wherein at least one of domains        (a), (b) and (c) is from a cutinase belonging to a different        species.        4. The isolated recombinant protein of any one of items 1 to 3,        wherein domain (a), (b), and/or (c) is from a multi-domain        cutinase belonging to Kineococcus radiotolerans, Cellulomonas        flavigena, Cellulomonas bogoriensis, or Cellulomonas cellasea.        5. The isolated recombinant protein of any one of claims 1 to 4,        wherein:    -   (i) domain (a) is:        -   (1) a cutinase catalytic domain having at least 50%, at            least 55%, at least 60%, at least 65%, at least 70%, at            least 75%, at least 80%, at least 85%, at least 90%, or at            least 95% sequence identity to amino acid sequence of SEQ ID            NOs: 2, 30, 33, 36, 39, or 42-45, or to the cutinase            catalytic domain of SEQ ID NO: 1, 13-20, or 25;        -   (2) a cutinase catalytic domain comprising or consisting of            the amino acid sequence of SEQ ID NOs: 2, 30, 33, 36, 39, or            42-45, or the cutinase catalytic domain of SEQ ID NO: 1,            13-20, or 25; or        -   (3) both (1) and (2);    -   (ii) domain (b) is:        -   (1) a threonine/proline-rich linker domain having at least            50%, at least 55%, at least 60%, at least 65%, at least 70%,            at least 75%, at least 80%, at least 85%, at least 90%, or            at least 95% sequence identity to SEQ ID NO: 3, 31, 34, 37,            or 40, or to the threonine/proline-rich linker domain of SEQ            ID NO: 1 or 13-15;        -   (2) a threonine/proline-rich linker domain comprising or            consisting of the amino acid sequence of SEQ ID NO: 3, 31,            34, 37, or 40, or to the threonine/proline-rich linker            domain of SEQ ID NO: 1 or 13-15;        -   (3) a threonine/proline-rich linker domain having at            threonine/proline content of at least 50%, at least 55%, at            least 60%, at least 65%, at least 70%, at least 75%, at            least 80%, at least 85%, at least 90%, or at least 95% over            a stretch of 15 to 55 consecutive amino acids; or        -   (4) any combination of (1) to (3);    -   (iii) domain (c) is:        -   (1) a polymer binding domain having at least 50%, at least            55%, at least 60%, at least 65%, at least 70%, at least 75%,            at least 80%, at least 85%, at least 90%, or at least 95%            sequence identity to SEQ ID NO: 4, 32, 35, 38, or 41;        -   (2) a polymer binding domain comprising or consisting of the            amino acid sequence of SEQ ID NO: 4, 32, 35, 38, or 41, or            to the polymer binding domain of SEQ ID NO: 1 or 13-15; or        -   (3) both (1) and (2); or    -   (iv) any combination of (i) to (iii).        6. The isolated recombinant protein of any one of items 1 to 5,        wherein said recombinant protein:    -   (i) is produced fused to a heterologous carrier protein or        heterologous tag that enables secretion of said recombinant        protein, and/or facilitates purification or detection of said        recombinant protein;    -   (ii) is truncated from a native N terminal signal sequence found        on a corresponding native cutinase; or    -   (iii) both (i) and (ii).        7. The isolated recombinant protein of item 6, wherein said        carrier protein is YebF as defined by SEQ ID NO: 8.        8. A polynucleotide molecule comprising a nucleic acid sequence        encoding the recombinant protein as defined in any one of items        1 to 7, operably linked to a heterologous promoter.        9. The polynucleotide of item 8, wherein the recombinant protein        encoded by said polynucleotide: (i) lacks an N terminal signal        sequence of a native cutinase; (ii) is fused to a heterologous        carrier protein; or (iii) both (i) and (ii).        10. A vector comprising the polynucleotide molecule as defined        in item 8 or 9.        11. A cell comprising the isolated recombinant protein as        defined in any one of items 1 to 7, the polynucleotide molecule        as defined in item 8 or 9, or the vector as defined in item 9.        12. The cell of item 11, which is a bacterial cell, a yeast        cell, a non-yeast fungal cell, an insect cell, a plant cell, an        animal cell, or a mammalian cell.        13. The cell of item 12, wherein said cell is from E. coli,        Bacillus subtilis, or Streptomyces spp.        14. A composition comprising: (i) the isolated recombinant        protein as defined in any one of items 1 to 7; and (ii) a        bleaching agent and/or an organic solvent, wherein said        recombinant protein retains said cutinase activity when present        in said composition.        15. The composition of item 14, wherein:    -   (i) said bleaching agent is:        -   (1) an oxidizing agent which is or comprises: hydrogen            peroxide, a peroxide other than hydrogen peroxide, a            non-peroxide oxidizing agent, ozone, sodium percarbonate,            sodium perborate, or any combination thereof; and/or        -   (2) a reducing agent which is or comprises: sodium            dithionite, sodium dithionite, sulfur dioxide, a sulfite, a            bisulfite, a sodium borohydride, or any combination thereof;    -   (ii) said organic solvent which is or comprises: methanol,        2-propanol, or another alcohol, dimethyl sulfoxide (DMSO),        dimethylformamide (DMF), or acetone; or    -   (iii) both (i) and (ii).        16. The composition of item 14 or 15, wherein:    -   (i) said bleaching agent is present at a concentration of:        -   (1) at least 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm,            350 ppm, 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, 650            ppm, 700 ppm, 750 ppm, 800 ppm, 850 ppm, 900 ppm, 950 ppm,            1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm,            4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm,            or 7000 ppm; or        -   (2) between 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350            ppm, 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, 650 ppm,            700 ppm, 750 ppm, 800 ppm, 850 ppm, 900 ppm, 950 ppm, or            1000 ppm, and 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500            ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500            ppm, or 7000 ppm;    -   (ii) said organic solvent is present as a concentration of:        -   (1) at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,            20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v; or        -   (2) between 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,            20%, or 25%, and 30%, 35%, 40%, 45%, or 50% v/v; or    -   (iii) both (i) and (ii).        17. The composition of any one of items 14 to 16, further        comprising: (i) an additional recombinant enzyme which is: a        lipase, an esterase, a pectate lyase, a pectinase, a cutinase, a        cellulase, a hemicellulose, an amylase, or any combination        thereof, or another recombinant enzyme; (ii) a detergent; (iii)        a surfactant; (iv) a buffer; (v) a chelator; or (vi) any        combination of (i) to (v).        18. The composition of any one of items 14 to 17, which is: (i)        a cleaning product, a degreaser, a disinfectant, a bleaching        product, or any combination thereof; or (ii) feed or a feed        additive.        19. Use of the isolated recombinant protein as defined in any        one of items 1 to 7, or the composition as defined in any one of        items 14 to 17, for: (i) cleaning, degreasing, disinfecting,        bleaching product, biofilm control, or any combination        thereof; (ii) for inactivating mycotoxin; (iii) bioscouring or        other treatment of fabrics or textiles; (iv) catalyzing        trans-esterification reactions; (v) vegetable oil extraction;        or (vi) enhanced oil recovery.        20. The use of item 18 for inactivating ochratoxin.        21. A method for producing the recombinant protein as defined in        any one of items 1 to 7, said method comprising: (i) culturing a        suitable host cell expressing a polynucleotide encoding said        recombinant protein, wherein said polynucleotide encodes a        truncated recombinant protein lacking an N terminal signal        sequence; and (ii) isolating said recombinant protein.        22. The method of item 21, wherein said polynucleotide is        operably linked to a heterologous promoter.        23. The method of item 21 or 22, wherein said polynucleotide        encodes said recombinant protein fused to a heterologous carrier        protein.

In other implementations, the present description relates to one or moreof the following items.

1. A recombinant protein having cutinase activity, said proteincomprising a cutinase catalytic domain that inactivates mycotoxin.2. The protein of item 1, wherein said cutinase catalytic domaininactivates ochratoxin.3. The protein of item 1 or 2, wherein said cutinase catalytic domaininactivates aflatoxin.4. The protein of any one of items 1 to 3, wherein said cutinasecatalytic domain inactivates ochratoxin and aflatoxin.5. The protein of any one of items 1 to 4, wherein said cutinasecatalytic domain shares at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% sequence identity to the cutinase catalyticdomain of any one of SEQ ID NOs: 2 and 13-20.6. A recombinant protein having cutinase activity, said proteincomprising a cutinase catalytic domain having at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% sequence identity tothe cutinase catalytic domain of SEQ ID NO: 2.7. The protein of any one of items 1 to 6, wherein said cutinasecatalytic domain shares at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% sequence identity to thecutinase catalytic domain of SEQ ID NO: 2.8. The protein of any one of items 1 to 6, wherein the cutinasecatalytic domain comprises the amino acid sequence of SEQ ID NO: 2 orthe cutinase catalytic domain of any one of SEQ ID NOs: 13-20.9. The protein of any one of items 1 to 8, further comprising a polymerbinding domain operably linked to said cutinase catalytic domain.10. The protein of item 9, wherein said polymer binding domain binds topoly(hydroxybutyrate), triacin, tributyrin, trimiristin, tripalmitin,polyvinyl acetate (PVA), polyethylene terephthalate (PET), orpolytrimethylene terephthalate (PTT).11. The protein of item 9 or 10, wherein said polymer binding domaincomprises an amino acid sequence sharing at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% sequence identity to thepolymer binding domain of any one of SEQ ID NOs: 4 and 13-15.12. The protein of item 11, wherein said polymer binding domaincomprises the amino acid sequence of any one of SEQ ID NOs: 4 and 13-15.13. The protein of any one of items 1 to 12, wherein said cutinasedomain and said polymer binding domain are operably linked by a linkerdomain.14. The protein of item 11, wherein said linker domain is aproline/threonine-rich linker domain.15. The protein of item 13 or 14, wherein said linker domain comprisesan amino acid sequence sharing at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% sequence identity to: SEQ ID NO: 3; residues220-247 of SEQ ID NO: 13; residues 226-236 of SEQ ID NO: 14; or residues228-276 of SEQ ID NO: 15.16. A polynucleotide encoding the protein of any one of items 1 to 15.17. A vector comprising the polynucleotide of item 16 operably linked toa heterologous promoter.18. A cell comprising the vector of item 17.19. Use of the protein as defined in any one of items 1 to 15 forinactivating mycotoxin.20. The use of item 19, wherein said mycotoxin comprises ochratoxin.21. The use of item 19, wherein said mycotoxin comprises aflatoxin.22. The use of item 20, wherein said mycotoxin comprises ochratoxin andaflatoxin.23. Use of the protein of any one of items 1 to 15 for degrading apolymer.24. The use of item 23, wherein said polymer is a polyester.25. The use of item 23, wherein said polymer is: cutin; polycaprolactone(PCL); polylactic acid (PLA); poly(1,3-propylen adipate (PPA);poly(hydroxybutyrate); triacin; tributyrin; trimiristin; tripalmitin;polyvinyl acetate (PVA); polyethylene terephthalate (PET); orpolytrimethylene terephthalate (PTT).26. Use of the protein as defined in any one of items 1 to 15 for themanufacture of: a cleaner and/or degreaser; animal feed; a surfacetreatment product in the pulp and paper industries; or a product for thedegradation and/or control of biofilm.27. A kit for inactivating mycotoxins, said kit comprising the proteinas defined in any one of items 1 to 15; and a suitable container.28. A method for expressing a transgenic KrCUT protein in a cell, saidmethod comprising expression of a YebF-KrCUT nucleotide in the cell.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

Definitions

In the present description, a number of terms are extensively utilizedand they should be given the meaning of the person of skill in the artwith a mind willing to understand. Furthermore, in order to provide aclear and consistent understanding of the specification and claims,including the scope to be given to such terms, the following definitionsare provided.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. In general, the terminology “about” is meant to designate apossible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10% of a value is included in the term “about”.

As used herein, “polynucleotide” or “nucleic acid molecule” refers to apolymer of nucleotides and includes DNA (e.g., genomic DNA, cDNA), RNAmolecules (e.g., mRNA), and chimeras thereof. The nucleic acid moleculecan be obtained by cloning techniques or synthesized. DNA can bedouble-stranded or single-stranded (coding strand or non-coding strand[antisense]). Conventional deoxyribonucleic acid (DNA) and ribonucleicacid (RNA) are included in the terms “nucleic acid molecule” and“polynucleotide” as are analogs thereof (e.g., generated usingnucleotide analogs, e.g., inosine or phosphorothioate nucleotides). Suchnucleotide analogs can be used, for example, to prepare polynucleotidesthat have altered base-pairing abilities or increased resistance tonucleases. A nucleic acid may comprise only conventional sugars, basesand linkages, as found in RNA and DNA, or may include both conventionalcomponents and substitutions.

As used herein, the term “gene” refers to nucleic acid molecules whichmay be isolated from chromosomal DNA, and very often include an openreading frame encoding a protein, e.g., polypeptides of the presentinvention. A gene may include coding sequences, non-coding sequences,introns and/or regulatory sequences, as well known.

As used herein, “recombinant protein” refers to a protein produced usingcells that do not have, in their native state, an endogenous copy of theDNA able to express the protein. The cells produce the recombinantprotein because they have been genetically altered by the introductionof the appropriate isolated nucleic acid sequence. The term“recombinant” also includes reference to a cell, or nucleic acid, orvector, that has been modified by the introduction of a heterologous(exogenous or foreign) nucleic acid or the alteration of a nativenucleic acid to a form not native to that cell, or that the cell isderived from a cell so modified. Thus, for example, recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell, express mutants of genes that are found within thenative form, or express native genes that are otherwise abnormallyexpressed, under-expressed or not expressed at all.

As used herein, the terms “hybridizing” and “hybridizes” are intended todescribe conditions for hybridization and washing under which nucleotidesequences at least about 60%, at least about 70%, at least about 80%,more preferably at least about 85%, even more preferably at least about90%, more preferably at least 95%, more preferably at least 98% or morepreferably at least 99% homologous to each other typically remainhybridized to each other. A preferred, non-limiting example of suchhybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in 1×SSC,0.1% SDS at 50° C., preferably at 55° C., preferably at 60° C. and evenmore preferably at 65° C. Highly stringent conditions include, forexample, hybridizing at 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDSand washing in 0.2×SSC/0.1% SDS at room temperature. Alternatively,washing may be performed at 42° C. The skilled artisan will know whichconditions to apply for stringent and highly stringent hybridizationconditions.

The terms “identity” and “percent identity” are used interchangeablyherein. For the purpose of this invention, it is defined here that inorder to determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical positions/totalnumber of positions (i.e., overlapping positions)×100). Preferably, thetwo sequences are the same length. Thus, In accordance with the presentinvention, the term “identical” or “percent identity” in the context oftwo or more nucleic acid or amino acid sequences, refers to two or moresequences or subsequences that are the same, or that have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., 60% or 65% identity, preferably, 70-95% identity, more preferablyat least 95% identity), when compared and aligned for maximumcorrespondence over a window of comparison, or over a designated regionas measured using a sequence comparison algorithm as known in the art,or by manual alignment and visual inspection. Sequences having, forexample, 60% to 95% or greater sequence identity are considered to besubstantially identical over their full lengths.

In a related manner, the terms “homology” or “percent homology”, referto a similarity between two polypeptide sequences, but take into accountchanges between amino acids (whether conservative or not). As well knownin the art, amino acids can be classified by charge, hydrophobicity,size, etc. It is also well known in the art that amino acid changes canbe conservative (e.g., they do not significantly affect, or not at all,the function of the protein). A multitude of conservative changes areknown in the art, Serine for threonine, isoleucine for leucine, argininefor lysine etc., Thus the term homology introduces evolutionisticnotions (e.g., pressure from evolution to a retain function of essentialor important regions of a sequence, while enabling a certain drift ofless important regions).

An “isolated polynucleotide” or “isolated nucleic acid molecule” is anucleic acid molecule (DNA or RNA) that is not immediately contiguouswith both of the coding sequences with which it is immediatelycontiguous (one on the 5′ end and one on the 3′ end) in the naturallyoccurring genome of the organism from which it is derived.

As used herein, an “isolated polypeptide” or “isolated protein” isintended to include a polypeptide or protein removed from its nativeenvironment.

The term “variant” refers herein to a polypeptide, which issubstantially similar in structure (e.g., amino acid sequence) to apolypeptide disclosed herein or encoded by a nucleic acid sequencedisclosed herein without being identical thereto. Thus, two moleculescan be considered as variants even though their primary, secondary,tertiary or quaternary structures are not identical. A variant cancomprise an insertion, substitution, or deletion of one or more aminoacids as compared to its corresponding native protein. A variant cancomprise additional modifications (e.g., post-translationalmodifications).

As used herein, the term “mycotoxin” refers to any toxic product formedin a mold, fungus, and/or yeast that exhibits significant toxicity to ahuman or animal when ingested or otherwise contacted (e.g., on the skin,in the lungs (inhaled), in the eyes, etc.). Thus, specificallycontemplated mycotoxins include aflatoxins (and particularly B1, B2, G1,and G2), fumonisins (and particularly B1, B2, and B3), ochratoxin,deoxynivalenol (DON, vomitoxin), T-2 toxin, and zearalenone.

As used herein the expressions “inactivates”, “inactivating”,detoxifies”, or “detoxifying”, in the context of mycotoxins, refers tothe processing of a mycotoxin in accordance with the present inventionsuch that an undesirable effect of the mycotoxin is reduced oreliminated. In some embodiments, such undesirable effects may includereduction or elimination of toxicity to a human or animal when theprocessed mycotoxin is ingested or otherwise contacted (e.g., on theskin, in the lungs (inhaled), in the eyes, etc.).

Other definitions may be provided throughout the description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 is a schematic representation of the domain organization of KrCUTand sequence characteristics of some conserved regions. SP, signalpeptide; TIP-rich, threonine-praline rich linker; “KrCUT206” (containing206 amino acids) is a truncated version of KrCUT lacking thepolymer-binding domain. “KrCUT189” (containing 189 amino acids) is atruncated version of KrCUT206 further lacking the T/P-rich linker. Inset(a): “cutinase motifs” containing the predicted catalytic triads(S114-D184-H198) and conserved cysteines (C103, C180, C187) according toWang et al., 2010. The sequences shown in panel (a) are represented inthe Sequence Listing as follows:

-   -   Kr SEQ ID NO: 49 and SEQ ID NO: 50;    -   Cf SEQ ID NO: 51 and SEQ ID NO: 52;    -   Fs SEQ ID NO: 53 and SEQ ID NO: 54;    -   Pi SEQ ID NO: 55 and SEQ ID NO: 56;    -   Rh SEQ ID NO: 57 and SEQ ID NO:58;    -   Mg SEQ ID NO: 59 and SEQ ID NO: 60;    -   Mf SEQ ID NO: 61 and SEQ ID NO: 62.

Inset (b): Homology to substrate-binding domains of PH8 depolymerases.The sequences shown in panel (b) are represented in the Sequence Listingas follows:

-   -   Kr SEQ ID NO: 63;    -   Cf SEQ ID NO: 64;    -   Pl SEQ ID NO: 65;    -   Ts SEQ ID NO: 66;    -   Ct SEQ ID NO: 67;    -   Tf SEQ ID NO: 68;    -   Nd SEQ ID NO: 69.        Abbreviations of bacterial source: Cf, Cellulomonas flavigena        DSM 20109 (ZP_04367703); Ct, Comamonas testosteroni (BAA22882);        Fs, Fusarium solani (P00590); Kr, Kineococcus radiotolerans        cutinase (YP_001363838); Mf, Monilinia fructicola (AAZ95012);        Mg, Magnaporthe grisae (CAA43717); Nd, Nocardiopsis dassonvillei        subsp PHB depolymerase (ZP_04334644); Pi, Phytophthora infestans        (AAY43367); PI, Paucimonas lemoignei (AAB48166); Rh, Rhodococcus        (jostii) RHA1 (YP_700622). Tf, Thermobifida fusca YX PHB        depolymerase (YP_288143, re-annotated as WP_011290529.1); and        Ts, Thermobifida sp. BCC23166 PHB depolymerase (ACF17837).

FIG. 2 shows purification of recombinant K. radiotolerans cutinase andtruncated derivatives. Proteins were analyzed by 10% SDS PAGE, andsilver stained. The molecular weight markers are indicated alongside;(a) lane 1, fraction from SP-Sepharose™ showing YebF-KrCUT fusionprotein (39 kDa) and mature KrCUT (31 kDa); lanes 2 and 3 from HiLoad™Superdex™ 75 showing the separated protein bands; (b) lane 4,YebF-KrCUT189 (33 kDa) from HiLoad™ Superdex™ 75.

FIG. 3 shows substrate specificity of full-length KrCUT and KrCUT189.Activity (U/mg) towards selected p-nitrophenol esters are compared.

FIG. 4 shows pH optima curves for activity of KrCUT and KrCUT189.

FIG. 5 shows thermostability curves for KrCUT. Activity of the enzymeafter incubation at the defined temperatures and for the selected timeperiods was measured.

FIG. 6 shows the enzymatic activity of protein purification elutionfractions corresponding to KrCUT (fractions C1-C10) and YebF-KrCUT(fractions D9-D11).

FIG. 7 shows GC analysis of cutin monomers released by KrCUT. Thenumbers on the peaks correspond to the retention times of the releasedproducts as described in the text.

FIG. 8 shows degradation of synthetic polyesters by YebF-KrCUT andKrCUT189. Enzyme concentration is 2.5 μM, unless otherwise stated. (A)Degradation of PCL films (▪, YebF-KrCUT; ▴, YebF-KrCUT, 1.25 μM; and □,KrCUT189), and PCL pellets (, YebF-KrCUT; and ∘, KrCUT189). The curveof PCL film degradation by 5 μM YebF-KrCUT approximated that of 2.5 μMenzyme concentration (not shown). (B) Degradation of PLA (, YebF-KrCUT;and ∘, KrCUT189) and polypropylene adipate (▪, YebF-KrCUT; and □,KrCUT189).

FIG. 9 shows adsorption isotherm of cutinase and PHB granules at 25° C.Purified KrCUT of concentrations ranging from 0.16 to 3.2 mg/mL weremixed with 25 mg PHB in 50 mM sodium phosphate (pH 8.0) in a totalvolume of 1 mL and mixed by gently shaking for 3 h. The PHB polymer wasremoved by centrifugation and the concentration of protein in thesupernatant was determined by the BCA method. The concentration of boundprotein at a particular concentration of cutinase was calculated as thedifference between a control without added PHB and free cutinase afterincubation with PHB.

FIG. 10 shows amounts of pectin released from natural hemp fibres bypectate lyase (PL) or in combination with cutinase (Cut) at differentconcentrations. The shaded bars indicate the two different incubationperiods (1.5 h and 3 h).

FIGS. 11 (A) and (B) show light microscopic analysis of hemp fibreobtained by enzymatic retting. (A) pectate lyase treatment, fibers areembedded into pectin and wax material; (B) pectate lyase and cutinasetreatment, fiber, F cell in bundles. (C) and (D) represents images fromscanning electron microscopy (SEM): (C) pectate lyase; (D) pectate lyaseand cutinase.

FIG. 12 shows the long term stability of recombinant KrCUT instabilisation buffer.

FIG. 13 shows examples of purified recombinant proteins separated bySDS-PAGE. Panel (A) shows the enzyme KrCUT, while panel (B) shows thevariant “Am-Kr” alone or fused to the heterologous carrier protein, YebF(“YebF-Am-Kr”).

FIG. 14 shows CD spectroscopy measurements in order to monitor theunfolding/refolding of various recombinant proteins: (A) KrCUT; (B)Am-Kr; (C); CfCUT; and (D) Cf-TP.

FIG. 15 shows a multiple sequence alignment (A) and a phylogenic tree(B) of native multi-domain cutinases from Kineococcus radiotolerans,Cellulomonas bogoriensis, Cellulomonas cellasea, and Cellulomonasflavigena.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable formentitled Sequence_Listing.txt, created Jan. 28, 2016 having a size of 84kb. The computer readable form is incorporated herein by reference.

SEQ ID NO: 1 ssKrCUT amino acid sequence SEQ ID NO: 2 KrCUT189 aminoacid sequence (cutinase catalytic domain of KrCUT) SEQ ID NO: 3 T/P-richlinker of KrCUT amino acid sequence SEQ ID NO: 4 Polymer binding domainof KrCUT amino acid sequence SEQ ID NO: 5 DNA encoding KrCUT optimizedfor expression in E. coli SEQ ID NO: 6 KrCUT amino acid sequence,lacking residues 1-33 of ssKrCUT SEQ ID NO: 7 DNA encoding YebF SEQ IDNO: 8 YebF amino acid sequence SEQ ID NO: 9 DNA encoding YebF-KrCUT SEQID NO: YebF-KrCUT amino acid 10 sequence SEQ ID NO: DNA encodingYebF-KrCUT189 11 SEQ ID NO: YebF-KrCUT189 amino acid 12 sequence SEQ IDNO: ssCfCUT: cutinase from Cf amino 13 acid sequence SEQ ID NO:ssCbCUT1: cutinase from Cb 14 amino acid sequence SEQ ID NO: ssCcCUT:cutinase from Cc 15 amino acid sequence SEQ ID NO: ssAmCUT: cutinasefrom Am 16 ATCC 39116 amino acid sequence SEQ ID NO: ssFrCUT: cutinasefrom Fr amino 17 acid sequence SEQ ID NO: ssAcCUT: cutinase from Ac 43118 amino acid sequence SEQ ID NO: ssCaCUT: cutinase from Ca 19 aminoacid sequence SEQ ID NO: ssRhCUT: cutinase from Rh 20 amino acidsequence SEQ ID NO: CfKr fusion protein amino acid 21 sequence SEQ IDNO: DNA encoding CfKr fusion 22 protein SEQ ID NO: KrCf fusion proteinamino acid 23 sequence SEQ ID NO: DNA encoding KrCf fusion 24 proteinSEQ ID NO: ssCbCUT2: cutinase from Cb 25 69B4 (KGM14008.1) amino acidsequence SEQ ID NO: AmCUT: cutinase from Am 26 ATCC 39116 amino acidsequence SEQ ID NO: Am-Kr: variant amino acid 27 sequence SEQ ID NO:CfCUT: cutinase from Cf 28 amino acid sequence SEQ ID NO: Cf-TP: variantamino acid 29 sequence SEQ ID NO: cutinase catalytic domain of 30 CfCUTamino acid sequence SEQ ID NO: T/P-rich linker domain of 31 CfCUT aminoacid sequence SEQ ID NO: polymer binding domain of 32 CfCUT amino acidsequence SEQ ID NO: cutinase catalytic domain of 33 CbCUT1 (KGM14009.1)amino acid sequence SEQ ID NO: linker of CbCUT1 34 (KGM14009.1) aminoacid sequence SEQ ID NO: polymer binding domain of 35 CbCUT1(KGM14009.1) amino acid sequence SEQ ID NO: cutinase catalytic domain of36 CbCUT2 (KGM14008.1) amino acid sequence SEQ ID NO: T/P-rich linker ofCbCUT2 37 (KGM14008.1) amino acid sequence SEQ ID NO: polymer bindingdomain of 38 CbCUT2 (KGM14008.1) amino acid sequence SEQ ID NO: cutinasecatalytic domain of 39 CcCUT (DSM 20118) amino acid sequence SEQ ID NO:T/P-rich linker of CcCUT 40 (DSM 20118) amino acid sequence SEQ ID NO:polymer binding domain of 41 CcCUT (DSM 20118) amino acid sequence SEQID NO: cutinase catalytic domain of 42 FrCUT (BMG5.12) amino acidsequence SEQ ID NO: cutinase catalytic domain of 43 AcCUT (431) aminoacid sequence SEQ ID NO: cutinase catalytic domain of 44 CaCUT (DSM44712) amino acid sequence SEQ ID NO: cutinase catalytic domain of 45RhCUT amino acid sequence SEQ ID NO: CbCUT2: cutinase from Cb 46(KGM14008.1) amino acid sequence SEQ ID NO: CcCUT: cutinase from 47CcCUT (DSM 20118) amino acid sequence SEQ ID NO: CbCUT1: cutinase from48 CbCUT1 (KGM14009.1) amino acid sequence ss = native N-terminal signalsequence Kr = Kineococcus radiotolerans Cf = Cellulomonas flavigena Cb =Cellulomonas bogoriensis Cc = Cellulomonas cellasea Am = Amycolatopsissp. Fr = Frankia sp. BMG5.12 Ac = Actinoplanes missouriensis Ca =Cryptosporangium arvum Rh = Rhodococcus TP = threonine/proline-richlinker domain from Kr

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Recombinant Proteins

In one aspect, the present description relates to an isolatedmulti-domain recombinant protein having cutinase activity. Themulti-domain recombinant protein may comprise: (a) a cutinase catalyticdomain; (b) a proline/threonine-rich linker domain positioned C-terminalof domain (a); and (c) a polymer binding domain positioned C-terminal ofdomain (b).

As used herein, the expression “cutinase catalytic domain” refers to apolypeptide domain that has the ability to hydrolyze cutin (and/oranother polymer substrate of interest hydrolyzable by the cutinasecatalytic domain) in a modular fashion (i.e., when present within alarger recombinant polypeptide of the present invention).

In some embodiments, the cutinase catalytic domain may be: (1) acutinase catalytic domain having at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% sequence identity of SEQ ID NOs: 2,30, 33, 36, 39, or 42-45; (2) a cutinase catalytic domain comprising theamino acid sequence of SEQ ID NOs: 2, 30, 33, 36, 39, or 42-45; or (3)both (1) and (2).

In some embodiments, the cutinase catalytic domain may share at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% sequenceidentity to the cutinase catalytic domain of any one of SEQ ID NOs: 2and 13-21.

As used herein, the expression “threoninelproline-rich linker domain” or“prolinelthreonine-rich linker domain” (T/P-rich linker domain) refersto a polypeptide domain that shares functional and/or structuralsimilarity to T/P-rich linker domains found in native multi-domaincutinases, such as those described herein from Kineococcus radiotolerans(e.g., accession ABS05574.1), Cellulomonas flavigena (e.g., accessionADG75999.1), Cellulomonas bogoriensis (e.g., accessions KGM14008.1), andCellulomonas cellasea (e.g., accession KGM03336.1).

In some embodiments, the T/P-rich linker domain may be athreonine/proline-rich linker domain having at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:3, 31, 34, 37, or 40. In some embodiments, the T/P-rich linker domainmay be a T/P-rich linker domain comprising the amino acid sequence ofSEQ ID NO: 3, 31, 34, 37, or 40.

In some embodiments, the T/P-rich linker domain may be a polypeptidedomain which has at least 20%, at least 25%, at least 30%, at least 35%,at least 40%, at least 50%, at least 55%, at least 60%, or at least 65%of its residues being a proline or a threonine, over a stretch of 15 to55 consecutive amino acids.

In some embodiments, the linker domain may comprise an amino acidsequence sharing at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% sequence identity to: SEQ ID NO: 3; residues 220-247 of SEQID NO: 13; residues 226-236 of SEQ ID NO: 14; or residues 228-276 of SEQID NO: 15.

As used herein, the expression “polymer binding domain” refers to aprotein domain that may have the ability to bind in a modular fashion(i.e., when present within a larger recombinant polypeptide of thepresent description) to a given polymer of interest (e.g., a polyesterhydrolyzable by a cutinase catalytic domain of the present description).

In some embodiments, the polymer binding domain may have at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% sequence identityto SEQ ID NO: 4, 32, 35, 38, or 41. In some embodiments, the polymerbinding domain may comprise the amino acid sequence of SEQ ID NO: 4, 32,35, 38, or 41.

In some embodiments, the polymer binding domain of a polypeptide of thepresent description may bind to poly(hydroxybutyrate), triacin,tributyrin, trimiristin, tripalmitin, polyvinyl acetate (PVA),polyethylene terephthalate (PET), or polytrimethylene terephthalate(PTT). In some embodiments, the polymer binding domain may comprise anamino acid sequence sharing at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% sequence identity to the polymer bindingdomain of any one of SEQ ID NOs: 4 and 13-15. In some embodiments, thepolymer binding domain comprises the amino acid sequence of any one ofSEQ ID NOs: 4 and 13-15.

In some embodiments, an isolated recombinant protein of the presentdescription may be a chimeric recombinant protein, wherein at least oneof domains (a), (b) and (c) is from a cutinase belonging to a differentspecies. Native multi-domain cutinases can be found in species such asKineococcus radiotolerans (e.g., accession ABS05574.1), Cellulomonasflavigena (e.g., accession ADG75999.1), Cellulomonas bogoriensis (e.g.,accessions KGM14008.1 and KGM14009.1), or Cellulomonas cellasea (e.g.,accession KGM03336.1).

In some embodiments, the above mentioned protein further comprises apolymer binding domain operably linked to the cutinase catalytic domain.As used herein, the expression “operably linked” refers to the covalentlinkage of two or more modular protein domains (e.g., a cutinasecatalytic domain and a polymer binding domain) such that each of themodular protein domains at least substantially retains its enzymaticactivity. Two or more protein domains that cooperate synergisticallywhen covalently linked is also within the meaning of “operably linked”as used herein.

In some embodiments, the above mentioned cutinase domain and the abovementioned polymer binding domain are operably linked by a linker domain.In some embodiments, the linker domain is 5 to 60, 5 to 50, 5 to 40, 5to 30, 5 to 20, 10 to 60, 10 to 50, or 10 to 40 amino acids in length.In some embodiments, the linker domain is a proline/threonine-richlinker domain. As used herein, the expression “prolinelthreonine-rich”refers to a polypeptide domain which has at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, or at least 65% of its residues being a proline or athreonine. In some embodiments, the linker domain comprises an aminoacid sequence sharing at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% sequence identity to: SEQ ID NO: 3; residues220-247 of SEQ ID NO: 13; residues 226-236 of SEQ ID NO: 14; or residues228-276 of SEQ ID NO: 15.

In another aspect, the present invention relates to isolated recombinantproteins sharing a minimum threshold of amino acid sequence identitywith any one of the polypeptides as described herein. In specificembodiments, the present invention relates to isolated polypeptideshaving at least 60%, 65%, 70%, 71%, 72, 73%, 74%, 75%, 76%, 77%, 78%,79, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to anyone of the above-mentioned polypeptides. Other specific percentage unitsthat have not been specifically recited here for brevity arenevertheless considered within the scope of the present invention.

In some embodiments, the isolated recombinant protein of the presentdescription may have improved stability to a bleaching agent, ascompared to a native cutinase enzyme that comprises the correspondingcutinase catalytic domain. As used herein, a “bleaching agent” refers toan agent that lightens or whitens a substrate through chemical reaction.Bleaching reactions usually involve oxidative or reductive processesthat degrade color. As used in the present context, “increasedstability” refers to an enzyme that generally retains a higher relativelevel of enzymatic activity, or exhibits a lower relative loss ofenzymatic activity, after being exposed to increasing concentrations ofa given bleaching agent, as compared to a suitable control enzyme (e.g.,native enzyme).

In some embodiments, the bleaching agent may be an oxidizing agent or areducing agent. The oxidizing agent may include hydrogen peroxide, aperoxide other than hydrogen peroxide, a non-peroxide oxidizing agent,ozone, sodium percarbonate, sodium perborate, or any combinationthereof. The reducing agent may include sodium dithionite, sodiumdithionite, sulfur dioxide, a sulfite, a bisulfite, a sodiumborohydride, or any combination thereof.

In some embodiments, the isolated recombinant protein of the presentdescription retains activity in the presence of a chelator (e.g., EDTAor GLDA). In some embodiments, the isolated recombinant protein of thepresent description retains activity in the presence of EDTA at aconcentration of between 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%,and 10% w/v). In some embodiments, the isolated recombinant protein ofthe present description retains activity in the presence of GLDA at aconcentration between 0.5%, 1%, 2%, 3%, or 4%, and 5% w/v).

In some embodiments, the isolated recombinant protein of the presentdescription may have improved stability to an organic solvent (e.g.,methanol, 2-propanol, or another alcohol, dimethyl sulfoxide (DMSO),dimethylformamide (DMF), acetone, or any combination thereof), ascompared to a native cutinase enzyme that comprises the correspondingcutinase catalytic domain. As used in the present context, “increasedstability” refers to an enzyme that generally retains a higher relativelevel of enzymatic activity, or exhibits a lower relative loss ofenzymatic activity, after being exposed to increasing concentrations ofa given organic solvent, as compared to a suitable control enzyme (e.g.,native enzyme).

In some embodiments, the isolated recombinant protein of the presentdescription may exhibit a broader temperature activity range, ascompared to a native cutinase enzyme that comprises the correspondingcutinase catalytic domain. Such properties may increase the versatilityof an enzyme for industrial applications. As used in the presentcontext, “broader temperature activity range” refers to an enzyme thatgenerally retains a higher relative level of enzymatic activity, orexhibits a lower relative loss of enzymatic activity, when incubatedacross a broader range of reaction temperatures, as compared to asuitable control enzyme (e.g., native enzyme). This is in contrast to anenzyme that, for example, shows a maximal level of activity at a givenoptimal temperature, but exhibits a relatively sharp decrease inactivity when the reaction temperature is progressively shifted awayfrom that optimal temperature.

It will be appreciated that this disclosure embraces the degeneracy ofcodon usage as would be understood by one of ordinary skill in the artand as illustrated in the table below.

Codon Degeneracies Amino Codons Ala/A GCT, GCC, GCA, GCG Arg/R CGT, CGC,CGA, CGG, Asn/N AAT, AAC Asp/D GAT, GAC Cys/C TGT, TGC Gln/Q CAA, CAGGlu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/H CAT, CAC Ile/I ATT, ATC,ATA Leu/L TTA, TTG, CTT, CTC, CTA, Lys/K AAA, AAG Met/M ATG Phe/F TTT,TTC Pro/P CCT, CCC, CCA, CCG Ser/S TCT, TCC, TCA, TCG, AGT, Thr/T ACT,ACC, ACA, ACG Trp/W TGG Tyr/Y TAT, TAC Val/V GTT, GTC, GTA, GTG STARTATG STOP TAG, TGA, TAA

Conservative substitutions: Furthermore, it will be understood by oneskilled in the art that conservative substitutions may be made in theamino acid sequence of a polypeptide without disrupting the structure orfunction of the polypeptide. Conservative substitutions are accomplishedby the skilled artisan by substituting amino acids with similarhydrophobicity, polarity, and R-chain length for one another.Additionally, by comparing aligned sequences of homologous proteins fromdifferent species, conservative substitutions may be identified bylocating amino acid residues that have been mutated between specieswithout altering the basic functions of the encoded proteins. The tablebelow provides an exemplary list of conservative substitutions.

Conservative Substitutions Type of Amino

Substitutable Amino Acids Hydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, 

Sulphydryl Cys Aliphatic Val, Ile, Leu, Met Basic Lys, Arg, His AromaticPhe, Tyr, Trp

indicates data missing or illegible when filed

In another embodiment, the present invention relates to functionalvariants of the recombinant proteins as described herein. “Functionalvariants” refers to recombinant proteins differing from their parentrecombinant protein by one or more amino acid substitutions, deletions,and/or insertions, yet which at least retain or provide higherbiological activity as compared to their parent recombinant protein.Functional variants include functional fragments (i.e., biologicallyactive fragments) of any one of the recombinant polypeptides of epresent invention. Such fragments include fewer amino acids than thefull length protein from which they are derived, but exhibit at leastone biological activity of the corresponding full-length protein.

In another aspect, the present invention includes fusion proteinscomprising a polypeptide of the present invention or a functionalvariant thereof, which is operatively linked to one or more unrelatedpolypeptide (e.g., heterologous amino acid sequences). “Unrelatedpolypeptides” or “heterologous polypeptides” or “heterologous sequences”refer to polypeptides or sequences which are usually not present closeto or fused to one of the polypeptides of the present invention. Such“unrelated polypeptides” or “heterologous polypeptides” having aminoacid sequences corresponding to proteins which are not substantiallyhomologous to the polypeptide sequences disclosed herein. Such“unrelated polypeptides” can be derived from the same or a differentorganism. In one embodiment, a fusion protein of the present inventioncomprises at least two biologically active portions or domains ofpolypeptide sequences disclosed herein.

In another embodiment, a recombinant protein of the present inventioncan be fused to a protein which enables or facilitates recombinantprotein purification and/or detection. For example, a polypeptide of thepresent invention can be fused to a protein such as glutathioneS-transferase (GST), and the resulting fusion protein can then bepurified/detected through the high affinity of GST for glutathione. Inanother embodiment, a recombinant protein of the present invention canbe fused or otherwise operably linked to a carrier protein (e.g., YebF)which enables its extracellular secretion. Such a linkage can be madesuch the carrier protein is cleavable upon secretion of the recombinantprotein of the present invention.

Recovery and Purification

In another aspect, polypeptides of the present invention may be presentalone (e.g., in an isolated or purified form), within a compositionincluding a carrier (e.g., an enzymatic composition for carrying out anindustrial process), or in an appropriate host. In one embodiment,polypeptides of the present invention can be recovered and purified fromcell cultures (e.g., recombinant cell cultures) by methods known in theart. In another embodiment, high performance liquid chromatography(“HPLC”) can be employed for the purification.

In another aspect, polypeptides of the present invention includenaturally purified products, products of chemical synthetic procedures,and products produced by recombinant techniques from a prokaryotic oreukaryotic host, including, for example, bacterial, yeast, fungal,plant, insect, animal and mammalian cells. Depending on the hostemployed in a recombinant production procedure, the polypeptides of thepresent invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses.

Polynucleotides

In some aspects, the present description relates to polynucleotidesencoding the recombinant proteins described herein. In some embodiments,the recombinant protein encoded by said polynucleotide may lack an Nterminal signal sequence often found on native cutinases. Therecombinant truncation of such native N terminal signal sequences mayincrease the enzymatic activity, and thus the utility of the recombinantenzyme.

In some embodiments, the polynucleotides of the present description mayencode a recombinant protein described herein which is fused to aheterologous carrier protein (e.g., YebF) and/or a heterologous tag(e.g., a purification or detection tag). Such fusions may not onlyenable the proper expression and/or secretion of the recombinationprotein from a host production cell, but also may facilitate theautolysis of recombination proteins over time, which may result intruncated enzyme having higher activity and/or increased structuralstability.

In some embodiments, the polynucleotides described here may be fused toa heterologous promoter to improve their expression in a host productioncell.

In some aspects, the present invention relates to a polynucleotideencoding a recombinant protein as described herein. In some embodiments,the polynucleotide may be RNA or DNA (e.g., cDNA).

In another aspect, the present invention relates to isolatedpolynucleotides sharing a minimum threshold of nucleic acid sequenceidentity with any one of the polynucleotides as described herein. Inspecific embodiments, the present invention relates to isolatedpolynucleotides having at least 60%, 65%, 70%, 71%, 72, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequenceidentity to any one of the polynucleotides as described herein. Otherspecific percentage units that have not been specifically recited herefor brevity are nevertheless considered within the scope of the presentinvention. Polynucleotides having the aforementioned thresholds ofnucleic acid sequence identity can be created by introducing one or morenucleotide substitutions, additions or deletions into the codingnucleotide sequences of the present invention such that one or moreamino acid substitutions, deletions or insertions are introduced intothe encoded polypeptide. Such mutations may be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

In some aspects, the present invention relates to a vector comprising apolynucleotide as described herein operably linked to a heterologouspromoter. As used herein in the context of polynucleotides, the term“heterologous” refers to the combination of two or more polynucleotidemolecules when such a combination is not normally found in nature. Forexample, the two molecules may be derived from different species and/orthe two molecules may be derived from different genes, e.g. differentgenes from the same species or the same genes from different species. Apromoter is thus heterologous with respect to an operably linkedtranscribable polynucleotide molecule if such a combination is notnormally found in nature, i.e., that transcribable polynucleotidemolecule is not naturally occurring operably linked in combination withthat promoter molecule. In some embodiments, the promoter may be aconstitutively active promoter, or may be an inducible promoter. In someembodiments, the promoter may be a viral promoter, bacterial cellpromoter, a yeast cell promoter, a fungal cell promoter, an insect cellpromoter, an animal cell promoter, or a mammalian cell promoter. Aheterologous promoter may be a synthetic promoter that differs from anendogenous promoter by the addition, deletion or substitution of one ormore nucleotides.

Vectors

Another aspect of the invention pertains to vectors (e.g., expressionvectors), containing a polynucleotide encoding a polypeptide of thepresent invention. As used herein, the term “vector” includes a nucleicacid molecule capable of transporting another nucleic acid molecule towhich it has been linked.

Cells

In another aspect, the present description features cells, e.g.,transformed host cells or recombinant host cells that contain apolynucleotide or vector described herein. A “transformed cell” or“recombinant cell” is a cell into which (or into an ancestor of which)has been introduced a polynucleotide or vector of the presentdescription by means of recombinant DNA techniques. Both prokaryotic andeukaryotic cells may be included, e.g., a bacterial cell, a yeast cell,a non-yeast fungal cell (e.g., a filamentous fungus), an insect cell, aplant cell, an animal cell, or a mammalian cell. In some embodiments,the cells may be from E. coli, Bacillus subtilis, or Streptomyces spp.

Expression of Transonic Proteins

Several experiments were conducted trying to express KrCUT as well asother recombinant proteins in E. coli. Various vectors were tested withthe codon-optimized gene of krCUT including and excluding the enzyme'snatural signal sequence to express the protein intracellular. Extensivegrowth and induction conditions were tested but all experiments yieldedvery low or no expression of the protein.

However, it has presently been found that expression of the KrCUTprotein could be achieved by the fusion of KrCUT to the YebF protein.This strategy yielded reproducible protein expression and secretion.

The active proteins (confirmed by N-terminal sequencing) have aninsertion of two amino acids (leucine and glutamine) that have beenintroduced as linkers between YebF and the natural occurring KrCUT andKrCUT206 sequence, respectively. In the mature expressed protein, thosetwo amino acids are not cleaved off through the autocatalytic action ofthe enzyme but remain on the protein's N-terminus.

Monitoring the protein after production reveals a cleavage of theN-terminal fused YebF protein which also results in an increase inenzyme activity (about 5 times). At the same time this N-terminalprotein cleavage appears to be a slow process which is not related toprotease activity but rather to autocatalytic protein hydrolysis as itoccurs also in a purified protein solution.

Protein fractions after purification on SP-Sepharose™ contain KrCUT orother recombinant enzymes described herein with and without intactYebF-N-terminal fusion, which can be separated using size exclusionchromatography. Over time, the proteins undergo a change in the proteinpatterns by completely losing the fusion protein YebF. After storage forseveral weeks at 4° C. The only protein band left is KrCUT (or otherrecombinant proteins lacking the YebF carrier protein) showing a singleprotein band which consists now exclusively of the truncated recombinantprotein.

Uses & Compositions

In some aspects, the present description relates to a compositioncomprising (i) the isolated recombinant protein as defined herein; and(ii) an additive. As used herein, an “additive” refers to an agent that,when added to a composition of the present description, increasesperformance, stability, and/or shelf-life, as compared to a compositioncomprising the recombinant protein alone.

In some aspects, the present description relates to a compositioncomprising (i) the isolated recombinant protein as defined herein; and(ii) a bleaching agent and/or an organic solvent, wherein saidrecombinant protein retains said cutinase activity when present in saidcomposition.

In some embodiments, the composition may be a “ready to use” formulationwherein the bleaching agent may be present at a concentration of atleast 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 400 ppm, 450ppm, 500 ppm, 550 ppm, 600 ppm, 650 ppm, 700 ppm, 750 ppm, 800 ppm, 850ppm, 900 ppm, 950 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm,3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, or7000 ppm. In some embodiments, the bleaching agent may be present at aconcentration of between 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm,350 ppm, 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, 650 ppm, 700 ppm,750 ppm, 800 ppm, 850 ppm, 900 ppm, 950 ppm, or 1000 ppm, and 1500 ppm,2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm,5500 ppm, 6000 ppm, 6500 ppm, or 7000 ppm.

In some embodiments, the composition may be a dilutable “concentrate”wherein the bleaching agent may be present at a concentration of atleast 3%, 4%, 5%, or 6% v/v.

In some embodiments, the bleaching agent may be an oxidizing agent or areducing agent. The oxidizing agent may include hydrogen peroxide, aperoxide other than hydrogen peroxide, a non-peroxide oxidizing agent,ozone, sodium percarbonate, sodium perborate, or any combinationthereof. The reducing agent may include sodium dithionite, sodiumdithionite, sulfur dioxide, a sulfite, a bisulfite, a sodiumborohydride, or any combination thereof.

In some embodiments, the organic solvent may be: methanol, 2-propanol,or another alcohol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF),or acetone. In some embodiments, the organic solvent may be present at aconcentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v. In some embodiments, theorganic solvent may be present at a concentration of between 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%, and 30%, 35%, 40%, 45%,or 50% v/v. In some embodiments, the organic solvent is DMSO and ispresent at a maximum concentration of 50% v/v. In some embodiments, theorganic solvent is acetone and is present at a maximum concentration of30% v/v.

In some embodiments, the compositions of the present description maycomprise one or more an additional recombinant enzyme(s). Examples ofsuch additional recombinant enzymes may include: a lipase, an esterase,a pectate lyase, a pectinase, a cutinase, a cellulase, a hemicellulose,an amylase, or any combination thereof. In some embodiments, thecomposition does not comprise a protease that degrades or inactivates arecombinant protein as defined herein. In some embodiments, thecomposition comprises a protease and a stabilizer (e.g., a polyol and/orboric acid).

In some embodiments, the compositions of the present description maycomprise one or more additives such as a detergent; a surfactant; abuffer; a chelator (e.g., EDTA or GLDA); a stabilizer; or anycombination thereof. In some embodiments, the composition comprises EDTA(ethylenediaminetetraacetic acid) at a concentration of between 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%, and 10% w/v. In some embodiments,the composition comprises GLDA (L-Glutamic acid N,N-Diacetic acid Tetrasodium) at a concentration between 0.5%, 1%, 2%, 3%, or 4%, and 5% w/v).In some embodiments, the composition comprises GLDA at a concentrationof at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/v). Insome embodiments, the composition comprises GLDA at a concentration ofat least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/v).

In some embodiments, the compositions may comprise an additive which isa stabilization buffer comprising one or more of sodium gluconate (e.g.,1-5% v/v), sodium carbonate (e.g., 1-5% v/v), sodium citrate (e.g., 1-5%v/v), and propylene glycol (e.g., 1-5% v/v). In some embodiments, thecompositions may comprise a stabilization buffer comprising one or moreof a polyol such as propylene glycol and glycerol, boric acid and boronderivatives, and sodium formate. In some embodiments, the compositioncomprises a polyol and/or boric acid. In some embodiments, thecompositions of the present description may be: a cleaning product, adegreaser, a disinfectant, a bleaching product, or any combinationthereof; or feed or a feed additive.

In some embodiments, the present description relates to the use of theisolated recombinant protein as defined herein, or the composition asdefined herein, for: (i) cleaning, degreasing, disinfecting, bleaching,biofilm control, or any combination thereof; (ii) for inactivatingmycotoxin (e.g., ochratoxin and/or aflatoxin); (iii) bioscouring orother treatment of fabrics or textiles; (iv) catalyzingtrans-esterification reactions; (v) vegetable oil extraction; or (vi)enhanced oil recovery.

In some embodiments, the present description relates to the isolatedrecombinant protein as defined herein, or the composition as definedherein, for use in: (i) cleaning, degreasing, disinfecting, bleaching,biofilm control, or any combination thereof; (ii) for inactivatingmycotoxin; (iii) bioscouring or other treatment of fabrics or textiles;(iv) catalyzing trans-esterification reactions; (v) vegetable oilextraction; or (vi) enhanced oil recovery.

In some embodiments, the present description relates to the use of theisolated recombinant protein as defined herein, or the composition asdefined herein, for the manufacture of an industrial product for: (i)cleaning, degreasing, disinfecting, bleaching, biofilm control, or anycombination thereof; (ii) for inactivating mycotoxin; (iii) bioscouringor other treatment of fabrics or textiles; (iv) catalyzingtrans-esterification reactions; (v) vegetable oil extraction; or (vi)enhanced oil recovery.

In some embodiments, the present description relates to a kit comprisinga recombinant protein as defined herein and a suitable container. Insome embodiments, the kit may be for one or more of the above mentioneduses.

In some aspects, the present invention relates to the use of arecombinant protein as described herein for inactivating mycotoxin.

In some aspects, polypeptides of the present invention may be useful formycotoxin detoxification (mycotoxin inactivation). Advantageously, andunlike some other fungal cutinases that may have been used for mycotoxindetoxification, polypeptides of the present invention may be useful forinactivating more than one mycotoxin. In some embodiments, polypeptidesof the present invention may be useful for inactivating Ochratoxin. Insome embodiments, polypeptides of the present invention may be usefulfor inactivating Aflatoxin. In some embodiments, polypeptides of thepresent invention may be useful for inactivating both Ochratoxin andAflatoxin (e.g., from the fungi strains Aspergillus flavus and/orAspergillus niger).

In some aspects, the present invention relates to a kit for inactivatingmycotoxins, said kit comprising a recombinant protein of the presentinvention as defined herein; and a suitable container.

In some aspects, the present invention relates to the use of arecombinant protein as described herein for degrading a polymer. In someembodiments the polymer is a homopolymer or a heteropolymer. In someembodiments, the polymer is a polyester. In some embodiments, thepolymer is: cutin; polycaprolactone (PCL); polylactic acid (PLA);poly(1,3-propylen adipate (PPA); poly(hydroxybutyrate); triacin;tributyrin; trimiristin; tripalmitin; polyvinyl acetate (PVA);polyethylene terephthalate (PET); or polytrimethylene terephthalate(PTT).

In other embodiments, polypeptides of the present invention may beemployed with one or more other enzymes (e.g., pectate lyase) for thedegradation/hydrolysis of a polymer of interest.

Applications of the recombinant proteins of the present invention inindustries such as food, laundry/detergent, textiles, recycling, and/orpolymer manufacturing are also envisioned. In some aspects, the presentinvention may relate to the use of a recombinant protein as describedherein in cleaners/degreasers (industrial and/or domestic); animal feed;the treatment of surface in the pulp and paper industries; and/or in thedegradation/control of biofilm.

The present invention is illustrated in further details by the followingnon-limiting examples.

Example 1 Material and Methods 1.1 Reagents and Chemicals.

p-Nitrophenyl (pNP)-esters were purchased from Fluka (Steinheim,Germany) or from Sigma-Aldrich (USA). Lactic acid, 6-hydroxyhexanoicacid, adipic acid, polycaprolactone (PCL; M_(n)=42500; M_(w)=65000),polylactic acid (PLA; M_(n)=30000; M_(w)=60000), poly(1,3-propylenadipate) (PPA; M_(n)=42500; M_(w)=4800), and poly(R)-3-hydroxybutyrate(PHB) were purchased from Sigma-Aldrich, Fluka or Alfa Aesar (Ward Hill,USA). Restriction endonucleases were from New England Biolabs(Pickering, ON, Canada), T4 DNA ligase was from Roche (Mannheim,Germany) and Titanium DNA polymerase from Clontech (Takara Bio company,USA).

1.2 Protein Expression (Small Scale).

A single colony of E. coli JM109 cells harboring either YebF-KrCUT orYebF-KrCUT189 was grown at 30° C. on a rotary shaker at 200 rpm in 10 mLTerrific Broth (TB) medium supplemented with 100 μg/ml ampicillin. Theovernight culture was used to inoculate 1 L fresh TB medium, then grownto OD₆₀₀ 0.4-0.5 under the same culture conditions and induced with 0.1mM IPTG for 16 h. The cells were collected by centrifugation at 6000 rpmfor 30 min (Beckman centrifuge, Model J2-21M) and the supernatant wasused as a source of cutinase. Protein concentration was determined usingthe bicinchoninic acid (BCA) method (Smith et al., 1985).

1.3 Fermentation in DASGIP System (Large Scale Protein Expression).

A single colony of E. coli JM109 cells harboring either YebF-KrCUT orYebF-KrCUT189 were cultured in LB-media containing ampicillin (0.1 g/L)and incubated at 30° C. in a rotary shaker at 200 rpm overnight. Thepreculture (5% v/v of the total fermentation volume) was added to TBmedia (1 L) in a 2 L fermenter (DASGIP, Germany). The temperature wasset to 30° C. and the pH was controlled at 7.0 by the addition ofconcentrated ammonium hydroxide. The cell broth was aerated at 1.0 vvmand stirred at 400-900 rpm to maintain 30% dissolved oxygen. Ifnecessary, the airflow was supplemented with oxygen. At an OD₆₀₀ of 9(˜7 h), cutinase expression was induced by the addition of IPTG (finalconcentration 1 mM); the temperature was set to 25° C., and glucose (33%w/w aqueous solution) was fed at an initial rate of 3 mL/h and graduallyincreased to 6 mL/h. The cells were harvested after 16 h andcentrifugation of the cell broth (6000 rpm, 4° C., 20 min) yielded 0.82to 0.87 L of supernatant that was used to purify the correspondingcutinase.

1.4 Protein Purification.

All purification procedures were performed at 4° C. on an ÄKTAexplorer™100 Air chromatography system (GE Healthcare). Culture supernatantcontaining active enzyme was concentrated and dialyzed against sodiumphosphate buffer (20 mM, pH 7.0) by cross flow filtration using theQuixStand™ system (GE Healthcare) equipped with a hollow fibre cartridge(3 kDa cut-off). The enzyme solution was loaded on a SP-Sepharose™ FFcolumn (XK 50/12) previously equilibrated with 20 mM sodium phosphatebuffer (pH 6.0). The flow rate was 4.0 mL/min. The column was washedwith the same buffer until no protein could be detected in the flowthrough, and the enzyme subsequently eluted with a linear gradient of0-0.6 M NaCl. Active fractions were pooled and concentrated byultrafiltration (membrane exclusion size 10 kDa, in a 100 mL stirringcell (Amicon, USA) and then applied to a HiLoad™ Superdex 75 prep grade(16/60) column previously equilibrated with 20 mM sodium phosphatebuffer (pH 7.0) containing 150 mM NaCl. Protein was eluted with the samebuffer at a flow rate of 1.5 mL/min and collected in 2 mL fractions. Theprotein profile was monitored by its absorbance at 280 nm.

1.5 Protein Sequencing.

Purified enzymes were separated by SDS-PAGE and transferred onto apolyvinylidene difluoride membrane (BioRAD, USA). N-terminal sequencingwas carried out by the Sheldon Biotechnology Centre (McGill University,Montreal, Canada). Phenylthiohydantoin amino acids were analyzed by HPLCusing a reversed-phase column.

1.6 Esterase Assay.

Esterase activity was determined by monitoring the formation of pNP at410 nm from various pNP-esters using a Beckman UV spectrophotometer(model DU 640) (21). Substrates were dissolved in isopropanol and wereadded to 50 mM sodium phosphate buffer pH 8.0 to give 10% (v/v) totalisopropanol concentration in a 1 mL reaction volume. pNP-butyrate wasused at a final concentration of 1.2 mM. For pNP-caprylate, myristateand palmitate, the final concentration ranged from 0.1 to 0.5 mM. Thereaction (1 mL) was started by adding an appropriate amount of enzyme.One enzyme unit (U) is defined as the amount of enzyme that produced 1μmol of product (pNP) per min; specific activity is expressed as U/mgprotein. A molar extinction coefficient of 15 mM⁻¹ cm⁻¹ for pNP is usedfor calculating enzyme activity.

1.7 Determination of Cutinolytic Activity.

Apple (Golden Delicious) cutin was used as substrate. The outer layer ofthe fruit was peeled by hand and cutin preparation carried out asdescribed by Gerard et al. (1993) with minor modifications. Discs ofapple peels were incubated at 50° C. in oxalate buffer [(oxalic acid (4g/L)/ammonium oxalate (16 g/L)] for 16 h and then collected and washedseveral times with deionized water. The sample was treated with 0.5%Novozymes Aspergillus niger pectinase (Sigma P2736) in 100 mM phosphatecitrate buffer pH 5.8 for 16 h. The cutin sheets were washed withethanol, followed by several washes with chloroform, and then incubatedin chloroform at 45° C. for 16 h, and finally dried to constant weight.The cutin was ground in a Cyclone sample Mill (UDY Corporation, FortCollings, Colo., USA).

A typical reaction mixture (2 mL) contained 20 mg cutin and anappropriate amount of cutinase in 25 mM sodium phosphate buffer, pH 8.0.The mixture was incubated in a water bath at 37° C. with gentle shakingfor 16 h. Assays were conducted in duplicate. At the end of thereaction, the remaining cutin was removed by centrifugation (10 min,5000 rpm). The resulting solution was acidified with hydrochloric acidand cutin monomers extracted with chloroform/methanol, according toBligh and Dyer (1959). The organic phase was removed by evaporationunder an air stream and the dried residue dissolved in 1 mLchloroform/methanol (85:15) and used for further derivatization.Trimethylsilylation of cutin monomers was performed with BSTFA [N,O-bis(trimethylsilyl) trifluoroactamide] and TMCS(trimethylchlorosilane) provided by the supplier in the ratio 99:1(Supelco, Bellefonte, USA) at 70° C. for 3 h, and the composition of themixture determined by GC-MS.

1.8 Analysis of Cutin Monomers by GC-MS.

Cutin monomers were identified by GC-MS on an Agilent 6890 gaschromatography system coupled to a 5973 quadrupole mass spectrometer. 1μL was injected under splitless condition on a 50 m×0.20 mm×0.33 μmDB-5MS capillary column (Agilent). The oven temperature program was 70°C. (for 1.39 min) to 136° C. at 64° C./min and to 290° C. at 2.5° C./min(for 55 min). Helium was used as carrier gas. The temperature at theinjection port was 250° C. The detector was run in El mode at 70 eV.Masses were scanned between 50 and 650 amu.

Components were identified by comparison of the MS spectra of theirtrimethylsilyl (TMSi) derivatives, as methyl ester TMSi ether or as TMSiester TMSi ether, with published spectra (Kallio et al., 2006; Philippset al., 2002).

1.9 HPLC-Analysis of Monomers after Polymer Degradation.

HPLC analysis was carried out on a Waters system consisting of a model600 pump and 717 Plus autosampler, equipped with a refractive indexdetector (Waters, model 2414) or PDA detector (model 2996). Organicacids were analyzed with a Transgenomic ICSep IC-ION-300 (300 mm×7.8 mmOD) column using 0.01N H₂SO₄ as mobile phase at a flow rate of 0.4mL/min at 35° C. Retention times are as follows: lactic acid, 23.2 min;6-hydroyhexanoic acid, 45.2 min; and adipic acid, 33.4 min.

1.10 Determination of Polymer Degradation by Size ExclusionChromatography.

Size exclusion chromatography (SEC) was performed using amulti-detection system from Viscotek (Houston, Tex.) consisting of aModel 302 Triple Detector Platform, including a refractive indexdetector, a four capillary viscometer and a 2 angles laser lightscattering detector, and a GPCmax Integrated pump, autosampler, anddegasser. Molar masses of the samples were determined with universalcalibration with the refractive index detector and the viscositydetector. The universal calibration curve was based on polystyrenestandards [Polymer Laboratories (Amherst, Mass.) and Aldrich (Oakville,ON)] using the molar masses determined by the manufacturer and theintrinsic viscosities measured by the apparatus. The software OmniSEC™software from Viscotek was used for data collection and calibration.Separation in THF was performed by injecting 100 μL of 1.5-2 mg/mLsolutions of standards or samples into thermostatically controlledSuperRes™ columns [35° C.; PAS-102, PAS102.5, PAS-103L, each 300 mm×8mm; PolyAnalytik (London, ON)]. The flow rate was 1 mL/min.

1.11 Degradation of Polyesters.

An appropriate amount of cutinase was added to 20 mg of the polymer tobe analyzed (PCL, PLA, PHB, and PPA) in 50 mM phosphate buffer, pH 8.The reaction mixture (2 mL) was shaken at 37° C. at 175 rpm on anorbital shaker. Samples were taken after 1, 3, 6, and 24 h and aliquotsof 50 μL were analyzed by HPLC; the remaining mixtures were frozen andsubsequently lyophilised. The resulting solid (˜40-50 mg) was dissolvedin THF, filtered, diluted (1.5-2 mg/mL), and analyzed by SEC.

Alternatively, a thin film was prepared from 1 g of commercial availablePCL pellets and used for degradation. The polymer was dissolved indichloromethane in a 1 L beaker. The solvent was allowed to evaporateovernight and the resulting film dried to constant weight under reducedpressure. Prior to enzymatic treatment the film was cut into pieces (20mg) of approximately 1.5 cm×1.5 cm and thickness of approximately 0.1mm. The reaction mixture was monitored by HPLC and SEC as describedabove.

1.12 Action of KrCUT on Plant Material.

Hemp stems (50 cm, 25 g) were treated enzymatically with KrCUT (100 U/g)together with a cloned pectate lyase (20 U/g) described by Xiao et al.(2008). The reaction was carried out in 50 mM Tris-HCl, pH 8.0supplemented with 0.5 mM CaCl₂ (required for pectate lyase). The sampleswere incubated at 37° C. with gentle shaking at 100 rpm for 16 h. Thestems were removed from the enzymatic solution, peeled off by hand, andthe resulting fibre washed with hot water to inactivate the enzymes.After removal of the outer layer debris by rinsing with cold water, thefibres were air-dried. A sample of the hemp stem treated only withpectate lyase was prepared for comparison. These experiments wereconducted in duplicate. The dried fibres were spread onto a microscopeslide and observed by light microscope. A scanning electron microscopy(SEM) analysis was also conducted using a Hitachi S-4700 at a voltage of2 kV to observe the fibre surface and also fiber bundle.

1.13 Effect of KrCUT in Removing Pectin from Hemp Fibre.

Hemp fibres (German variety, 150 mg) were treated with either pectatelyase (PL, 10 U) or PL combined with various amounts of KrCUT (40, 60and 100 U). Control experiments containing no enzyme or 100 U of KrCUTwere conducted for comparison. After incubation at 37° C., aliquots ofthe reaction mixture were taken at 1 h 30 min and 3 h, and analyzed forpectin degradation products using the thio-barbituric acid (TBA) method(52) which detects released unsaturated compounds spectrophotometricallyat 550 nm.

1.14 Adsorption Assays.

Adsorption assays were carried out following procedures of Kasuya et al.(1999). Purified KrCUT and KrCUT189 at concentrations ranging from 0.16to 3.2 mg/mL were added to a suspension of 25 mg PHB in 50 mM sodiumphosphate (pH 8.0) in a total volume 1 mL. The mixture was incubated atroom temperature with gentle shaking for at least 3 h. The PHB polymerwas removed by centrifugation and the concentration of protein in thesupernatant was determined by the BCA method. The concentration of boundprotein at a particular concentration of cutinase was calculated as thedifference between a control without added PHB and free cutinase afterincubation with PHB. An isotherm of [Bound] (mg/mg PHB) vs [Free] (mM)was generated and binding parameters were determined by non-linearregression using the Langmuir equation:

$E_{ad} = {E_{\max}\left( \frac{{K\lbrack E\rbrack}e}{1 + {{K\lbrack E\rbrack}e}} \right)}$

where [E]=E_(ad)+[E]e, is the concentration of the protein added;E_(max) is the maximum amount of protein adsorbed on the polyestergranules, and K is the adsorption equilibrium constant of the protein.

Example 2 Sequence and Phylogenetic Analyses of Cutinase from K.radiotolerans

The 885-bp Krad_4111 locus tag of the K. radiotolerans genome(YP_001363838.1; Copeland, 2007) was selected as the result of a BLASTsearch using the amino acid sequence of FsCUT as a query sequence. Apolypeptide having 294 amino acids corresponding to the full lengthsequence of a cutinase from K. radiotolerans was identified. Abioinformatic analysis of the 294-amino acid sequence of full lengthKrCUT (hereinafter referred to as “ssKrCUT”; SEQ ID NO: 1) revealed amodular domain organization consisting of a 33-amino-acid signal peptide(amino acids 1-33 of SEQ ID NO: 1), a catalytic domain (amino acids34-222 of SEQ ID NO: 1; SEQ ID NO: 2), and unlike other characterizedcutinases, the presence of a C-terminal extension made up of aPro/Thr-rich linker (amino acids 223-239 of SEQ ID NO: 1; SEQ ID NO: 3),and a designated polymer-binding domain (amino acids 240-294 of SEQ IDNO: 1; SEQ ID NO: 4) (FIG. 1).

Example 3 Cloning of Cutinase-Encoding Gene and Variants

A synthetic gene encoding full-length cutinase from K. radiotoleransidentified in Example 2 but lacking the 33-amino acid endogenousN-terminal signal sequence was constructed after codon optimization bythe reduction of G+C content from 77.2% to 59% for its expression in E.coli. The synthetic gene having the nucleotide sequence set forth in SEQID NO: 5 was cloned in a recombinant pUC57 plasmid provided by GenScriptCorporation (Piscataway, N.J. USA). The specific primers, P1:5′-GAGCTGCAGGCGACTTGCAGTGACGTC-3′ and P2 5′-GCAGAAGCTTTCAACAAGCAGTTACCAAG-3′, were then designed to amplify the mature portion ofthe KrCUT-encoding gene (amino acids 34-294 of SEQ ID NO: 1; SEQ ID NO:6; herein referred to as “KrCUT”); P1 and P3: 5′-TGTAAGCTTTCATGTGGTAGGAACCGGCGT-3′ were used to amplify a truncated version KrCUT206 (aminoacids 34-222 of SEQ ID NO: 1; SEQ ID NO: 2; herein referred to as“KrCUT189”) lacking the C-terminal 72 amino acids, which includes theT/P-rich linker as well as the polymer-binding domain. The indicatedprimer sets were designed to incorporate a 5′ PstI site and 3′ HindIIIsite (underlined) for directional cloning in the IPTG-inducible vectorpKK223-3. The PCR parameters were: 94° C. 3 min, 30 cycles of 94° C. for45 s, Tm-3° C. for 30 s, 68° C. for 1 min, followed by a final extensionat 68° C. for 10 min.

To express KrCUT and KrCUT189 in a secreted form, the E. coliextracellular carrier protein YebF (Zhang et al., 2006; SEQ ID NO: 8)was used to construct fusion proteins. The gene encoding YebF (366-bpminus stop codon but including the signal peptide; SEQ ID NO: 7) wasamplified by oligonucleotides P4:5′-CGAGGAATTCATGGAGAAAAACATGAAAAAAAG-3′ and P5:5′-GAACTGCAGACGCCGCTGATATTCCGC-3′, containing the EcoRI and PstIrestriction sites (underlined). The PCR fragment was gel purified andcloned upstream of the cutinase gene in pKK223-2 to producepKK223-3-YebF-KrCUT and pKK223-3-YebF-KrCUT189. These recombinantplasmids were transformed into E. coli JM109 cells by conventionaltechniques (Sambrook et al., 1989). The cloned genes were verified byDNA sequencing using a Big Dye™ DNA sequencing kit (Applied Biosystems)and an automated DNA sequencer (Model 377, ABI Prism). The nucleotidesequence encoding the YebF-KrCUT fusion protein is set forth in SEQ IDNO: 9 and the corresponding amino acid sequence of YebF-KrCUT is setforth in SEQ ID NO: 10. The nucleotide sequence encoding theYebF-KrCUT189 fusion protein is set forth in SEQ ID NO: 11 and thecorresponding amino acid sequence of YebF-KrCUT189 is set forth in SEQID NO: 12.

Example 4 Expression and Purification of YebF-KrCUT and YebF-KrCUT189Fusion Proteins

The YebF-KrCUT and YebF-KrCUT189 fusion proteins were expressed from E.coli as described in Example 1, sections 1.2 and 1.3.

Cutinase activity, measured using pNP-butyrate as substrate as describedin Example 1, section 1.6, was detected in E. coli [pKK233-2-YebF-KrCUTand pKK233-2-YebF-KrCUT189] culture 16 h after IPTG induction with themaximum activity found by 20 h (not shown).

Production of the 39-kDa YebF-KrCUT (FIG. 2A, lane 1) fusion protein wasconfirmed by SDS-PAGE analysis), consistent with the summed masses ofthe 10-kDa YebF carrier protein and the 29-kDa mature KrCUT. FIG. 2A,lane 1, resulted from one column purification step using theSP-Sepharose™ FF as described in Example 1, section 1.4. Production ofthe YebF-KrCUT189 fusion protein was also confirmed by SDS-PAGEanalysis.

The YebF-KrCUT fusion protein was further purified as described inExample 1, section 1.4 to electrophoretic homogeneity in two stepsinvolving cation exchange and size exclusion chromatography. Proteinsbound on the SP-Sepharose™ eluted in two active peaks: peak 1 (minor) ata lower ionic strength, and peak 2 (major) at higher ionic strength (notshown). The height of peak 1 eluting from the SP-Sepharose™ columnincreased over time while peak 2 decreased in height, indicating someunknown processing of peak 1 protein species under storage conditions(not shown). This was confirmed by SDS-PAGE analysis of fractions fromthe major peak 2, which showed two protein bands of M_(r) 39-kDa and31-kDa (for YebF-KrCUT). These two species could be separated by anadditional SEC step (FIG. 2, lanes 2 and 3). In FIG. 2A, lanes 2 and 3,the two bands from lane 1 were separated by loading the fraction of lane1 on a HiLoad Superdex™ 75 prep grade (16/60) column.

The YebF-KrCUT189 fusion protein, which also eluted in two active peaks,was purified to electrophoretic homogeneity the same way as forYebF-KrCUT. FIG. 2B, lane 4 represents YebF-KrCUT189 from HiLoad™Superdex™ 75 prep grade (16/60) column, the fraction was collected aftera first column using SP-Sepharose FF.

N-terminal amino acid sequencing performed as described in Example 1,section 1.5 helped to establish the identity of each of the proteinbands with respect to YebF-KrCUT and YebF-KrCUT189. The procedure isdescribed with respect to YebF-KrCUT in the following paragraph, but asimilar approach was used to establish the identity of each of theprotein bands for YebF-KrCUT189.

The sequence ANNETSKSVT derived from the 39 kDa protein band correspondsto the first 10 amino acids of the mature portion of YebF, confirmingprevious result that the carrier protein is cleaved immediately afterthe 21-amino acid secretory leader sequence (Zhang et al., 2006). TheN-terminal amino acid sequence (AT[A]SDVDWF) of the 31-kDa species isvirtually identical to the first ten amino acids of the mature cutinase.The discrepancy of Ala in the third position versus Cys in theDNA-predicted sequence is due to the fact that Cys without modificationis not detectable by Edman degradation.

The above analysis confirmed that when the YebF-KrCUT and YebF-KrCUT189proteins were purified immediately after fermentation, they exclusivelyyielded the intact YebF-KrCUT and YebF-KrCUT189 fusion proteins,respectively. Upon storage for several weeks at 4° C., the intact fusionproteins underwent processing to produce a lower molecular weightspecies corresponding to the cleavage of the YebF portion, resulting inthe polypeptides KrCUT and KrCUT189.

The presence of YebF appeared to greatly decrease the activity of thefusion proteins. For example, the processed KrCUT exhibited somethree-time higher specific activity compared to that of YebF-KrCUT (162versus 47 U/mg with pNP-palmitate as substrate). The results are shownin FIG. 6, in which fractions C1-C10 correspond to the first peak(KrCUT) and fractions D9-D11 correspond to the second peak (YebF-KrCUT).

Example 5 Substrate Range

The KrCUT189 protein was purified in a similar way as YebF-KrCUT asdescribed above (FIG. 2B) and its activity towards a number of p-NPesters was compared to those of KrCUT (FIG. 3). With KrCUT, the highestactivity was obtained with pNP butyrate showing a 8-fold higher activityover pNP-palmitate which is in accordance with the activity profile ofother cutinases (Kim et al., 2003). The KrCUT189 variant generally gavelower activity with various substrates compared to the full lengthprotein. The highest activity of KrCUT189 was towards pNP-myristate,which was surprisingly higher than that of KrCUT. In both cases, thereis little activity towards pNB-acetate.

Example 6 pH Optimum and Thermostability

Both KrCUT and KrCUT189 showed a pH optimum of 8 (FIG. 4). UniversalBuffer solutions were prepared according to Britton and Robinson(Surinenaite et al., 2002), having the selected pH value from 5-11.Activity of KrCUT and KrCUT189 were measured at the indicated pH, usingp-nitrophenyl butyrate as substrate as described in Example 1, section1.6). Curves were obtained by plotting values of activity against pH.

Enzyme stability was studied over the temperature range of 40-60° C.using purified KrCUT. Aliquots were withdrawn at defined times (10, 30and 80 min), chilled on ice, and assayed for activity against p-NPbutyrate at room temperature. KrCUT appeared thermostable up to 45° C.At 50° C. and above there was virtually no activity left after 10 min ofheat treatment (FIG. 5).

Example 7 Potential Inhibitors of Cutinase Activity

KrCUT cutinase activity was assayed as described in Example 1, section1.6 in the presence of various chemicals using p-NP palmitate assubstrate under standard assay conditions. The zinc ion dramaticallyinhibited cutinase activity with a complete loss of activity at 1 mM andover 90% at 0.1 mM. Mg²⁺ showed only 19% enzyme inhibition at 10 mM; and50% for Fe²⁺ at 1 mM. Cu²⁺ at 1.0 mM had little effect. The enzyme wasnot affected by the metal-chelating agent EDTA even at 10 mM. Theanionic surfactant SDS was a strong inhibitor of cutinase even at 0.01%retaining only 28% activity. 0.1% SDS resulted in a complete loss ofactivity. Organic solvents, e.g. alcohols exhibited a strong inhibitingeffect on cutinase in concentration above 15-20%. In particular, the2-propanol used to dissolve the substrate and that is present in thestandard assay at 10% (v/v), strongly reduces enzyme activity when itexceeds 15%. Dimethylsulfoxide (at 30%) and dimethylformide (at 5%)inhibited cutinase activity by about 70%. Interestingly,para-chloromercuribenzoate at concentrations 0.01 to 1 mM had nodiscernible effect on KrCUT implying that there is no free sulfhydrylgroup among the six cysteines found in KrCUT.

Example 8 Hydrolysis of Cutin

To measure cutinase activity, assays of cutin hydrolysis were performedas described in Example 1, section 1.7 at two different enzymeconcentrations. Characteristic cutin components, C₁₆ and C₁₈ hydroxylfatty acids, were identified by GC-MS analysis. One representative massspectrum of the enzymatic reaction is shown in FIG. 7.18-Hydroxy-octadeca-9,12-dienoic acid, 10,16-dihydroxyhexadecanoic acidand 9,10,18-trihydroxyoctadecanoic acid, with retention times of 55.13,55.92 and 64.20, respectively, were found to be the major products ofhydrolysis (Table 1).

TABLE 1 Momoneric products released from apple cutin by cloned K.radiotolerans cutinase. Retention 20 Units 200 Units time Cutinhydrolysis products Area (%) Area (%) 55.13 18-hydroxy-octadeca-9,12-18.0 ± 6.3  2.22 ± 0.13 dienoic acid 55.24 9,16-dihydroxyhexadecanoicn.d  3.81 ± 0.62 acid 55.92 10,16-dihydroxyhexadecanoic 39.25 ± 2.5 27.74 ± 0.41 acid 63.37 9,10,18-trihydroxyoctadec- n.d 13.25 ± 0.4010,12-dienoic acid 64.20 9,10,18-trihydroxyoctadecanoic n.d 25.55 ± 0.10acid 66.19 9,10,18-trihydroxyoctadec-9 n.d  6.25 ± 0.40 enoic acidHydrolysis was carried out at two different units of activity ofcutinase. Not detected (n.d)

Additional components of C16 and C18 fatty acids were identified as9,16-dihydroxyhexadecanoic acid (55.24 min),9,10,18-trihydroxyoctadec-10,12-dienoic acid (63.37 min), and9,10,18-trihydroxyoctadec-9 enoic acid (66.19 min).

Example 9 Degradation of Synthetic Polyesters by KrCUT and KrCut189

An initial plate clearing assay indicated that a lawn of E. coli JM109cells harboring YebF-KrCUT was capable of producing a large clearingzone with PCL as substrate (not shown). To quantify this further,pellets of polycaprolactone (PCL) as well as films of PCL were incubatedwith KrCUT at various enzyme concentrations (1.25 μM, 2.5 μM, and 5 μMrepresenting 4.7, 9.5 and 19 enzyme Units) at 37° C. The degradation ofthe polymer was followed over time by analyzing aliquots of the reactionmixture by HPLC for the detection of the released monomer(6-hydroxyhexanoic acid) and characterization of the remaining polymerby SEC after lypholization of the sample. KrCUT was able to releasenearly 90% of the acidic monomers with enzyme concentration of either2.5 or 5 μM in 6 hours. Even with a very low concentration of the enzyme(1.25 μM) thin films of PCL were nearly completely degraded after 24hours (FIG. 8A).

SEC analysis confirmed the results obtained by HPLC. Comparison of thepeak area corresponding to that of the polymer showed a gradual decreaseover time with complete disappearance of the polymer in experimentsconducted with 2.5-5 μM after 24 hours. This correlated well with theincrease in acid detected (Table 2).

TABLE 2 Evidence of PCL degradation: comparison of SEC and HPLC analysisM_(n) M_(w) time (h) PCL (SEC) (Daltons) (Daltons) acid (HPLC) 0 100% 44000 63300 0% 1 61% 53800 64400 28% 3 27% 55000 66600 60% 6 12% 5820068100 85% 24 0.3%  69500 74000 100%A dramatic increase in M_(n) and M_(w), from 44000 and 63300 to 69500and 74000 emphasized the preference of KrCUT for low molecular polymers.The degradation of PCL pellets proceeded more slowly with 18% of themaximum yield of 6-hydroxy hexanoic acid were detected in the reactionmixture after incubation with 2.5 μM of protein for 24 hours (FIG. 8A).The decrease in depolymerization is presumably due to the limitedsurface area of the PCL pellets compared to the films. Nearly identicalresults were obtained when PCL pellets and PCL films were incubated withKrCUT189 (2.5 μM) (FIG. 8A).

Both KrCUT and KrCUT189 (2.5 μM; 2 Units) only degraded approximately 5%of PLA (FIG. 8B). However, in the case of poly(1,3-propylene adipate), apolymer consisting of alternating diacid and diol subunits, 12% and 17%of adipic acid were found in the reaction mixture containing KrCUT andKrCUT189, respectively, after 24 hours (FIG. 8B). All in all, it appearsthat both enzymes exhibited a preference for longer chain monomers suchas 6-hydroxy hexanoic acid and adipic acid (both C6 unit in polymer)compared to lactic acid (C2 unit).

Example 10 Polymer-Binding Domain of KrCUT

Different polymers (cellulose, PLA, PCL, and PHB) were tested toevaluate the ability of KrCUT and KrCUT189 to bind these materials.Binding assays for PCL and PLA could not be performed since bothpolymers were degraded by the enzymes. KrCUT did not bind to cellulose,indicating that the binding domain of the cutinase is distinct from acellulose binding domain (CBD). On the other hand, KrCUT bindsefficiently to PHB but did not degrade. The KrCUT189 variant showed nobinding to PHB, indicating that essentiality of the C-terminal extensionfor this activity.

The kinetics of KrCUT adsorption to PHB is shown in FIG. 9. The curveshows the relation between the adsorbed cutinase and the equilibriumconcentration of the enzyme, with the adsorption to the PHB surfaceexpressed by the Langmuir adsorption equation. As a result, the maximumamount of cutinase absorbed on the PHB granules (Emax) was determined tobe 23.68±0.03 μg/mg, and the adsorption equilibrium constant (K) wascalculated to be 0.12±0.03 ml/mg PHB.

Example 11 Effect of KrCUT on Hemp Fibre

The quantity of pectin released from natural hemp fibre by KrCUT inconjunction with the action of a thermostable pectate lyase was assessedas described in Example 1, section 1.13. The thermostable pectate lyasealone released a certain amount of pectin as expected of thepectinolytic activity (Xiao et al., 2008). With an increasing amount ofadded cutinase, a higher percentage of pectin degradation products weredetected when the quantity of added pectate lyase was fixed. When KrCUTwas incubated with hemp fibre at the highest concentration in theabsence of pectate lyase, negligible amounts of pectin from the fibrematerial were detected as compared to a control reaction without anyadded enzyme (FIG. 10).

The effect of KrCUT on the hemp fibre integrity was visualized usinglight microscopy and SEM. Light microscopic images revealed thattreatment of fibres by both cutinase and pectate lyase appeared toproduce more separated fibre bundles, whereas pectate lyase alone showedmore fibres embedded in pectin and wax material (FIG. 11). The SEMpictures showed that cutinase treatment gave more separated fibreshaving a cleaner surface, compared to those of pectate lyase treatedsamples.

Example 12 Effect of KrCUT on Inactivation of Mycotoxin

Fungi strains Aspergillus flavus and Aspergillus niger were cultivatedand used for Aflatoxin and Ochratoxin isolation, respectively.

Reactions were performed in 2 mL volumes in Eppendorf tubes comprisingthe mycotoxin to be analyzed and 0.3 mg/mL KrCUT enzyme. Ochratoxin Awas added at a concentration of 200 ppb. The samples for Aflatoxincontain the B1 and B2 types of the mycotoxin in the concentration of 600ppb and 9 ppb, respectively. In control reactions, the enzyme volume wasreplaced by an equivalent amount of water. The reactions were incubatedat 37° C., samples were withdrawn at time periods 0, 1 h, 2 h, followedby the addition of equal volume of methanol 80% (v/v) for extraction.Reactions were stored, if needed until chromatographic analysis.

Analysis: samples were centrifugated and the supernatant analyzed forOchratoxin A and Aflatoxin by LC-MS/MS as described by Rudrabhatla etal., 2007. Results are shown in Tables 3 and 4.

TABLE 3 Effect of KrCUT on Ochratoxin Incubation time (h) ResidualOchratoxin % 0 100 1 56.63 2 0.68

TABLE 4 Effect of KrCUT on Aflatoxin mixture Residual Incubation time(h) Residual Aflatoxin B1 (%) Aflatoxin B2 (%) 0 100 100 3 48 48

The results in Tables 3 and 4 show that the KrCUT is able to act on bothaflatoxin and ochratoxin, in contrast to most other cutinases that canact only on one type of mycotoxin.

Example 13 Effect of KrCUT on Inactivation of Mycotoxin

Fungi strains Aspergillus flavus and Aspergillus niger were cultivatedand used for Aflatoxin and Ochratoxin isolation, respectively.

Reactions were performed in 2 mL volumes in Eppendorf tubes comprisingthe mycotoxin to be analyzed and 0.3 mg/mL enzyme. The samples forAflatoxin contain the B1 type of the mycotoxin in the concentration of1000 ppb. In control reactions, the enzyme volume was replaced by anequivalent amount of water. The reactions were incubated at 37° C.,samples were withdrawn at time periods 0, 1 h, 2 h, 4 h and 24 hfollowed by the addition of equal volume of methanol 80% (v/v) forextraction. Reactions were stored, if needed until chromatographicanalysis.

Analysis: samples were centrifugated and the supernatant analysed forOchratoxin A and Aflatoxin by LC-MS/MS as described by Rudrabhatla etal., 2007. Results are shown in Table 5.

TABLE 5 Effect of KrCUT on Aflatoxin Incubation time (h) ResidualAflatoxin B1 (%) 0 100 1 80 2 61.5 4 49 24 20

The results in Table 5 show that KrCUT is able to reduce amount of 1000ppb Aflatoxin B1 to 200 ppb within 24 h.

Example 14 Construction of Recombinant Fusion Polypeptides Having aCutinase Catalytic Domain Operably Linked to a Polymer-Binding Domain

Recombinant fusion polypeptides having a cutinase catalytic domainoperably linked to a polymer-binding domain may be constructed usingbasic recombinant DNA cloning techniques, making use of suitable domains(cutinase catalytic domains, linker domains, and polymerbinding-domains) from different cutinases. Examples of differentcutinases and the domains therein are shown in Table 6.

TABLE 6 Examples of different cutinases and the domains thereinCutinase amino acid sequence (signal  sequences are in lowercase letters; cutinase catalytic domains are in bold; Gene/proteinlinkers are highlighted in black; SEQ name/organismpolymer binding domains are underlined) ID NO: >GI:502883354mqvtptrrtlagyvaavaalasyttlatgasaAPACPDVEL 13 WP_013118330VFARGTGEAAGLGIVGRPLERALAAELPGRTVVATAVDYAA cutinaseSSSQASAGPGSGDMVAKVRSRAAACPGTQFVLGGYSQGATV [CellulomonasTDLALGIRTGVTAGTALPEDLAARVAAVVVYGNPLGLTRRT flavigena]IAQAAPAFATRTVEYCNAGDPVCEPGGGRFTAHITYATNGT VLEGARFAAARVTA

CVTASSLQHVRDGRAYPLWMRTYARGSGDPLGVLSSRTVVSLQADGTDTWRKVAAC >g|1697974256|gmsitpggrkvaaalittaaltstaaiatpataaapvagpva 14 b|KGM14009.1|tsTGDCPDVHVVFARGTGEPRGLGIVGRPFVSDLGDALPTM cutinaseTVTSYAVNYSANASQTSAGPGAGDMTSHVTSMAARCPGTQF [CellulomonasVLGGYSQGATVTSIAVGARSTSIRSRVLPANLEPRVAAVVV bogoriensisFGNPLGLTRRTIASEAPAYAAKSRDYCNRSDTVCGGRGDAR 69B4 = DSM

16987] VEADRAFRSVLRAYARGSRDPLGRLTSSDLVSLQRTGQDSW SVVPAC cutinasemstspvrrtltaallaaglaatsvalspaasatslpaaapl 25 [Cellulomonas sAGSCPDVQVVFARGTGERAGLGIIGRPFARALADELPGMT bogoriensisVTSHAVDYAAAASQRSAGPGATAMTDHVTAMAARCPGTQFV 69B4 = DSMLGGYSQGATVTSIALGIRAGTTTGRAIPDELSDRVAAVVVF 16987];GNPLGMRGQTIASASRTYADRAKDYCNSGDSICGRQPSTGR accessionGTHTGYATNGSTTDGARFAAGLVTA KGM14008.1

SDQVTCVTAQVSEHVEARRAIRGLTRAYARGTLEDIGRLRSTEEVSLRRSGTFSWTPTASC >gi|697886498|gmhqltppaapsrpraarrprrllaaalalaatsgalstlaa 15 b|KGM03336.1|ptavaAPACADVEVLFARGTGEAPGLGVLGTPFVRSVTSAL hypotheticalSDRTVTSYAVNYAAESSQRSAGPGATDLTNHLTATAAACPG proteinTRFVLGGYSQGATVVDLALGIRTGTTTGTAIPAALEPRVAA Q760_05675IVVFGNPLGISGRTIATASPTYAARARDFCATGDPVCGGGS [Cellulomonas 

cellasea DSM

20118] AGRAERRHGIAYATGSGDRIGWVSSFVRVSVQQTADGWERV LSC >gi|521090090|mvhnrlrkltllacavvglagtatvgtasgatsaaACSDLE 16 ref|WP_0204209IVFARGSGEAPGLGITGTPLVSDVKSALSSASVSSYAVDYA 95.1| cutinaseASYDQTSAGPGATDMSNHIKSTAAACPDTKFAIGGYSQGAS [AmycolatopsisVTDIAIGIRTYLGTGQTIPTELAPRVVAVIAFGNPLGLYGQ sp. ATCC 39116]TIKTASPTYGPKSLEFCNRGDNVCGGTGTGPGYGHLSYARDGSVDQAAAFIAKQYNAS >gi|648619273: 1−mrlarswkqravavfltavagtlgagitaspvsAATCSDVD 17 219 cutinase VVFARGSGELPGLGITGTPFVNSVKQGLTGKTVSSYAVNYA [Frankia sp.ADIAQTSDGAGATDMTRHVRSVAASCPNTKFVLGGYSQGAS BMG5.12]VTDISIGIRTFLGSGETIPTELAPRVAAVVVFGNPLALFGQKITTASPLYGPKAKEFCNLGDPVCAGGFNVFAHLTYGFDGS TANGASFAVSKVRA >gi|383779171|mnkrrkrnvatvgicaavvaagavitpqafagtlfggratt 18 ref|YP_00546373aAGDCSDVELVFARGTGEPQGLGIVGRPLARELAAALPDLA 7.1| putativeVGSFAVVYAAAGNQRSAGPGATNMSRHITEVAGECPDTRFV cutinaseIGGYSQGASVTDIAIGIRGAGTAGEAIPERLADRVAAVVVF [ActinoplanesGNPLGLQRRTIAGSSAVFGPKAKEFCNTGDPVCGGGGNFAA missouriensis HLAYPRNGSVQQAAAFAASKIAG 431] >gi|589395439|gmsvlltvrrvvtavaltatgltgiafaaspaTAACSDVQVV 19 b|EXG80196.1|FARGSTEAPGLGILGRPLVSAVQQQLPGLTVDSYAVDYAAN CutinaseVSQTSAGPGATDMSDHITEVAARCPDTEFVIGGYSQGASVT [CryptosporangiumDIAIGIRTTLGRGGTIPENLAPRIKAVTVFGNPLRLSRQTI arvum DSMNSASQLYGRKAIDICATGDPVCGNGANAAAHLRYAFDGSVT 44712]RAAQFAANLVRTT >gi|653334177| mkrrliaysiaalaisatavalptgvasAAPCSDVDVSFAR20 ref|WP_0274949 GTGELPGLGITGTPFVNSVKSQLSDRSVSTYAVNYAADFTQ 01.1|ASAGPGSRDLVAHLNSVAASCPSTKFVIGGYSQGATVVTNA MULTISPECIES:VGLRTPSSFTGAVIPAAIADRIEAVVVFGNPFGLTGRKIET cutinaseASSTYGSRTNSFCNFGDPVCQIGGFNTFAHLTYGTNGSTTQ [Rhodococcus] GASFAAAQVRS

Further examples of constructions of fusion proteins are shown below.

14.1 Fusion Protein CfKr

Amino acid sequences of this construct contain the catalytic domain ofcutinase from Cellulomonas flavigena (CfCUT) fused to the C-terminalregion (T/P-rich linker and polymer binding domain) from Kinecoccusradiotolerans cutinase. The amino acid sequence of this fusion proteinis set forth in SEQ ID NO: 21, in which residues 1-187 correspond to thecutinase catalytic domain from CfCUT, residues 188-206 correspond to theT/P-rich linker of KrCUT, and residues 207-262 correspond to the polymerbinding domain of KrCUT. To ensure extracellular secretion of thisfusion protein, the N-terminus thereof can be fused to the C-terminus ofYebF (SEQ ID NO: 8) to give YebF-CfKr encoded by the nucleotide sequenceof SEQ ID NO: 22.

14.2 Fusion Protein KrCf

Amino acid sequences of this construct contain the cutinase catalyticdomain of KrCUT (SEQ ID NO: 2) fused to the T/P-rich linker of KrCUT(SEQ ID NO: 3), which in turn is fused to polymer binding domain ofCfCUT. The amino acid sequence of this fusion protein is set forth inSEQ ID NO: 23, in which residues 1-189 correspond to the cutinasecatalytic domain from KrCUT, residues 190-206 correspond to the T/P-richlinker of KrCUT, and residues 207-262 correspond to the polymer bindingdomain of CfCUT. To ensure extracellular secretion of this fusionprotein, the N-terminus thereof can be fused to the C-terminus of YebF(SEQ ID NO: 8) to give YebF-KrCf encoded by the nucleotide sequence ofSEQ ID NO: 24.

Example 15 Long Term Stability of KrCUT in Culture Medium

Stability over time and/or with temperature represents one of the maindesirable properties of an enzyme, for their utilization as ingredientsin cleaning product formulation. Most enzymes tend to lose activity withthe time.

The KrCUT was routinely produced in the form of secreted fusion protein,YebF-KrCUT in culture medium. Culture medium having KrCUT activity wasstored at 4° C. without any additives. Activity was determinedperiodically by using p-nitrophenyl laurate as substrate, and reportedin Table 7.

TABLE 7 Storage stability of KrCUT in culture medium at 4° C. Months 0 34 17 19 Activity 3.0 ± 0.94 25.67 ± 0.94 17.13 ± 0.70 16.66 ± 2.47 21.94± (U/mL) 1.83

At room temperature, KrCUT in culture medium without additives retainsfull activity for a period of 4 months.

Example 16 Stability of KrCUT in Stabilisation Buffer

For stability testing, culture medium showing KrCUT activity, andcontaining 100 mg total protein was used. Stabilisation buffer composedof sodium gluconate 1-5%, sodium carbonate 1-5%, sodium citrate 1-5% andpropylene glycol 1-5% was prepared; 50 mL enzyme solution was added to200 mL of buffer and stored at room temperature, after mixing. Sampleswere taken at defined time periods and the enzymatic activity wasmeasured using p-nitrophenyl dodecanoate as substrate.

Activity loss could be observed only after 4 months, and up to 65% ofthe initial activity was present after 9 months at room temperature.

Data are reported in FIG. 12.

For most of enzymes, more complex mixtures with different ingredients athigher concentrations are used for stabilisation, namely polyol such aspropylene glycol and glycerol, boric acid and boron derivatives andsodium formate.

In some test cleaning product formulations, the KrCUT enzyme wascombined with a mix of proteases from Bacillus. The efficacy of thecleaning product was reduced, demonstrating the contribution of theKrCUT to the overall efficacy of the cleaning product. However, the lossof KrCUT activity could be avoided or substantially reduced by theadditional of stabilizers to the formulation (e.g., polyols and/or boricacid).

Example 17 Stability of KrCUT in the Presence of Metal Ions

Enzymes such as lipases, amylases, and proteases are widely used incleaning product formulations, and these types of enzymes are generallysensitive to the presence or absence of metal ions. For example, lipasesgenerally require calcium for optimal activity. Amylases and proteasesalso generally require metal ions for optimal activity.

For improved performance, chelating agents (namely EDTA) are often addedto cleaning products. These chelating agents sequester metal ions andthus may negatively impact enzyme activity (e.g, of lipases, amylases,and proteases).

Interestingly, activity of KrCUT is not affected by EDTA or otherchelating agents often found in cleaning products. More specifically,10% w/v of EDTA does not inhibit KrCUT activity (data not shown), andthe enzyme retains 100% activity in the presence of GLDA (L-Glutamicacid N,N-Diacetic acid Tetra sodium), at a concentration of 5% w/v.

More particularly, stability in the presence of hydrogen peroxide makesKrCUT an interesting candidate for cleaning products with disinfectionproperties.

Example 18 Construction and Expression of Recombinant Enzymes HavingCutinase Activity

The recombinant enzymes listed in Table 8 were constructed, expressedand purified for further characterization as generally described inExamples 3 and 4.

TABLE 8 Recombinant enzymes having cutinase activityAmino acid sequence (YebF carrier  proteins are in lower case letters;SEQ cutinase catalytic domains are in ID NO:bold; linkers are highlighted in black; (without Enzyme Organism(s)polymer-binding domains are underlined) YebF) YebF- Kineococcusmeknmkkrgaflglllvsacasvfaannetsks  6 KrCUT radiotoleransvtfpkcedldaagiaasvkrdyqqnrvarwadd qkivgqadpvawvslqdiqgkddkwsvpltvrgksadihyqvsvdckagmaeyqrrATCSDVDVVF ARGTGETPGLGVVGGPFVRSLTGELSDRTVTSHAVDYAASSSQASAGPGATAMSAHVREVAAACPS TRFVLGGYSQGATVTDIALGIRTGTTTGTPVPAELAGRVAAVVVFGNPLGLSGRTIATASSTYGPK SKDYCNSSDSVCGSAPKTGTGGHLSYASNGSTT

RDSTRDHVAADRAVSLYGRAYARGSRDSLGATS SYNVVSLQQVEGGWRLVTAC YebF-Amycolatopsis meknmkkrgaflglllvsacasvfaannetsks 26 AmCUT sp. ATCC 39116vtfpkcedldaagiaasvkrdyqqnrvarwadd qkivgqadpvawyslqdiqgkddkwsvpltvrgksadihyqvsvdckagmaeyqrrACSDLEIVFA RGSGEAPGLGITGTPLVSDVKSALSSASVSSYAVDYAASYDQTSAGPGATDMSNHIKSTAAACPDT KFAIGGYSQGASVTDIAIGIRTYLGTGQTIPTELAPRVVAVIAFGNPLGLYGQTIKTASPTYGPKS LEFCNRGDNVCGGTGTGPGYGHLSYARDGSVDQAAAFIAKQYNAS YebF- Amycolatopsis meknmkkrgaflglllvsacasvfaannetsks 27Am-Kr sp. ATCC 39116; vtfpkcedldaagiaasvkrdyqqnrvarwadd Kineococcusqkivgqadpvawvslqdiqgkddkwsvpltvrg radiotoleransksadihyqvsvdckagmaeyqrrACSDLEIVFA RGSGEAPGLGITGTPLVSDVKSALSSASVSSYAVDYAASYDQTSAGPGATDMSNHIKSTAAACPDT KFAIGGYSQGASVTDIAIGIRTYLGTGQTIPTELAPRVVAVIAFGNPLGLYGQTIKTASPTYGPKS LEFCNRGDNVCGGTGTGPGYGHLSYARDGSVDQ

RDSTRDHVAADRAVSLYGRAYARGSRDSLGATS SYNVVSLQQVEGGWRLVTAC YebF-Cellulomonas meknmkkrgaflglllvsacasvfaannetsks 28 CfCUT flavigenavtfpkcedldaagiaasvkrdyqqnrvarwadd qkivgqadpvawvslqdiqgkddkwsvpltvrgksadihyqvsvdckagmaeyqrrAPACPDVELV FARGTGEAAGLGIVGRPLERALAAELPGRTVVATAVDYAASSSQASAGPGSGDMVAKVRSRAAACP GTQFVLGGYSQGATVTDLALGIRTGVTAGTALPEDLAARVAAVVVYGNPLGLTRRTIAQAAPAFAT RTVEYCNAGDPVCEPGGGRFTAHITYATNGTVLEGARFAAARVTA

CVTASSLQHVRDGRAYPLWMRTYARG SGDPLGVLSSRTVVSLQADGTDTWRKVAAC YebF-Cellulomonas meknmkkrgaflglllvsacasvfaannetsks 29 Cf-TP flavigena;vtfpkcedldaagiaasvkrdyqqnrvarwadd Kineococcusqkivgqadpvawvslqdiqgkddkwsvpltvrg radiotoleransksadihyqvsvdckagmaeyqrrAPACPDVELV FARGTGEAAGLGIVGRPLERALAAELPGRTVVATAVDYAASSSQASAGPGSGDMVAKVRSRAAACP GTQFVLGGYSQGATVTDLALGIRTGVTAGTALPEDLAARVAAVVVYGNPLGLTRRTIAQAAPAFAT RTVEYCNAGDPVCEPGGGRFTAHITYATNGTVL

TASSLQHVRDGRAYPLWMRTYARGSGDPLGVLS SRTVVSLQADGTDTWRKVAAC

The constructed and expressed enzymes include the predicted maturewild-type cutinases from Kineococcus radiotolerans (“KrCUT”),Amycolatopsis sp. ATCC 39116 (“AmCUT”, which lacks a linker domain and apolymer binding domain), and Cellulomonas flavigena (“CfCUT”), as wellas the chimeric variants “YebF-Am-Kr” (having AmCUT fused to theT/P-rich linker domain and the polymer binding domain of KrCUT), and“YebF-Cf-TP” (having the cutinase catalytic domain of CfCUT, theT/P-rich linker domain of KrCUT, and the polymer binding domain ofCfCUT). The domain structures of each of the recombinant enzymes listedin Table 8 are summarized in Table 9 (following autolytic cleavage ofthe respective YebF carrier proteins).

TABLE 9 Summary of domain structure of recombinant enzymes havingcutinase activity Enzyme Cutinase catalytic Linker Polymer binding namedomain from: domain from: domain from: KrCUT Kineococcus KineococcusKineococcus radiotolerans radiotolerans radiotolerans AmCUTAmycolatopsis sp. ATCC n/a n/a 39116 Am-Kr Amycolatopsis sp. ATCCKineococcus Kineococcus 39116 radiotolerans radiotolerans CfCellulomonas flavigena Cellulomonas Cellulomonas flavigena flavigenaCf-TP Cellulomonas flavigena Kineococcus Cellulomonas radiotoleransflavigena

Constructions

To ensure expression of the recombinant enzymes, synthetic genesencoding fusion proteins consisting of the carrier protein YebF (357 bp)and the corresponding full-length cutinases lacking the nativeN-terminal signal sequences were constructed.

The optimized sequences for reduction of G+C content were provided byBiomatik USA. The nucleotide sequences were cloned into the NcoI andHindIII sites of the pTrc2Bd vector, a derivative of the plasmidpTrcHis2B, obtained by deletion of the His Tag. The recombinant plasmidswere transformed in the β-galactosidase-positive and lactose inducibleE. coli MG1655 strain.

The synthetic gene of Amycolatopsis sp. ATCC 39116, cutinase (AmCUT)corresponds to 564 bp, after optimization the G+C content was reducedfrom 71% to 55%.

The recombinant enzyme variant Am-Kr was constructed as a 786 bppolynucleotide consisting of 561 bp AmCUT and 225 bp of the C-terminalportion of KrCUT. Codon optimization led to G+C content reduction from68% to 56%.

The synthetic gene encoding CfCUT corresponded to an 812 bp nucleotidesequence, in which G+C content was reduced from 78% to 59% after codonoptimization.

The polynucleotide encoding the variant CfTP represented a DNA fragmentof 789 bp.

Production at Small Scale

Expression of the recombinant enzymes were performed by growing the E.coli MG1655 harboring the recombinant plasmids pTrc3Bd. Colonies of thecells on LB agar plates were selected and used to prepare pre-culturesconsisting of 5 mL TB media supplemented with ampicillin (50 μg/mL). Theculture was incubated at 37° C., on a rotary shaker at 200 rpm, for 16h. The overnight culture was used to inoculate 1 L of fresh TB media.Recombinant enzyme production was induced by addition of 10 g/L lactose,at optical density (OD) 0.5, cells were allow to grow for 24 h,collected by centrifugation at 4000 rpm, 20 min (Eppendorf Centrifuge5810R). The supernatant represents the source of cutinase for activitymeasurements.

Production of Cutinase Variants in Bioreactor

Fermentation was performed in a 30-Litre computer-controlled, in-situsterilizable bioreactor (INFOR HT Techfors), with a working volume of 20L. The bioreactor was filled with 18 L of TB culture medium andsterilized at 121° C. for 20 min. A regulation system was used tocontrol the temperature at 30° C. throughout the experiment. A pH of 7.0was maintained constant by using acidic H₂SO₄ and basic NaOH regulation.

The stirring speed was first set at 400 rpm and then linked to dissolvedoxygen (DO) concentration at 25% saturation. The airflow velocity wasset to 1 vvm. The bioreactor was inoculated with 5% volume of cultureunder aseptic conditions, using pre-culture. First pre-culture wasperformed in 5 mL TB media, supplemented by ampicillin (50 μg/mL), andincubated at 37° C., 16 h, using bacteria colonies from LB agar plate.This culture was used to inoculate the second pre-culture, in a 1 Lflask, TB medium, incubation under the same conditions.

Expression of recombinant enzymes was initiated at OD600 of 7, by addinglactose at a concentration of 10 g/L. The cultivation was allowed tocontinue for 24 h, after induction, fermentation liquid was collected bycentrifugation, 8000 rpm for 30 min (Beckman centrifuge, Model AvantiJ26 XPI).

The following crude culture supernatants were obtained:

-   -   KrCUT (˜8 L); 1.6 U/mL (pNP-dodecanoate);    -   AmCUT (˜8 L); 1.2 U/mL (pNP-dodecanoate);    -   Am-Kr (˜16 L); 0.5 U/mL (pNP-dodecanoate);    -   CfCUT (˜16 L); 0.5 U/mL (pNP-dodecanoate); and    -   Cf-TP (˜8 L); 0.6 U/mL (pNP-dodecanoate).

Purification

In a first step, all 5 enzymes were concentrated to about 0.5 L using a10 kD cut-off holo fiber membrane. During this step, the enzymecontaining solutions became much darker, probably due to mediumingredients that remained loosely bound to proteins contained in thesolution and thus did not pass the 10 kD barrier.

After concentration, each enzyme solution was subjected to dynamicdialysis on a SPECTRA/POR™ 500-016 HP-75 dialysis module, 10 kD, 6500cm². In this step, the individual protein concentration was keptconstant, but salt and medium components were removed gradually. Theenzyme-containing solution was dialyzed against 20 mM sodium phosphate,pH 6.0 until the conductivity of the solution was lower than 2 mS/cm².

Theoretical isoelectric points and the molecular masses of the enzymeswere calculated using their putative amino acid sequences. The resultsand properties of each of the enzyme variants are shown in Table 10.

TABLE 10 Properties of recombinant enzymes expressed 1 Absorbance Calc.1 Molar Unit [280 nm] Absorbance Predicted Length MW microgram =extinction corr. [280 nm] Isoelectric charge Enzyme (aa) (kD) (pmol)coefficient to (mg/mL) of 1 mg/mL point at pH 7 YebF- 393 39.5 25.3137920 1.04 0.96 8.81 6.13 KrCUT KrCUT 261 26.1 38.37 16650 1.57 0.648.59 3.07 YebF- 309 32.3 30.98 34550 0.93 1.07 6.98 −0.03 AmCUT AmCUT187 18.8 53.11 13280 1.42 0.71 5.20 −3.10 YebF- 383 40 25.0 44320 0.901.11 8.04 2.04 Am-Kr Am-Kr 261 26.6 37.65 23050 1.15 0.87 6.32 −1.03YebF- 393 40.6 24.60 42320 0.96 1.04 8.69 4.97 CfCUT CfCUT 271 27.236.77 21060 1.29 0.77 8.29 1.90 YebF- 384 39.9 25.06 42330 0.94 1.068.94 6.97 Cf-TP Cf-TP 262 26.5 37.80 21060 1.26 0.80 8.84 3.90

Based on their calculated molecular weights, their isoelectric points,and other properties, the enzymes YebF-KrCUT and KrCUT, YebF-CfCUT andCfCUT, as well as YebF-Cf-TP and Cf-TP, were all predicted to bind to acation exchanger (Capto™-S at a pH of 6.0 and a conductivity below 2mS/cm²). The enzyme YebF-AmCUT was predicted to weakly bind to thecation exchanger, but not the enzyme AmCUT. The enzyme Yeb-F—Am-Kr waspredicted to bind to the cation exchanger, whereas the enzyme Am-Kr wasconsidered to bind minimally.

Despite the generally positive binding assessment of the above enzymes(with exception of AmCUT) to a cation exchanger (Capto™-S andSP-Sepharose™ XL were tested) under the selected conditions (pH 6.0, 2mS/cm²), only KrCUT bound completely to the resin. CfCUT did not bind atall and appeared completely in the flow through, whereas active enzymefor AmCUT, Am-Kr, and Cf-TP were found in both the column flow throughand in bound fractions.

Bound fractions of each enzyme (with the exclusion of CfCUT) wereconcentrated using stirring cell with a cut-off membrane of 10 kD, andsubjected to size exclusion chromatography using a Hi-Load™ Superdex™ μg16/60 column, which also allowed the assessment of the native molecularmass of the enzymes based on their retention time.

For KrCUT separated on SP-Sepharose™ XL, two active fractions (A7/A8,and A11-B1; chromatogram on shown) were collected. Both fractions showedsimilar activity but, due to the higher yield, fraction A11-B1 wasselected for further studies and purified by size exclusionchromatography.

For AmCUT separated on Capto™-S, only a very small amount of activeenzyme bound to the column and was subsequently concentrated andsubjected to size exclusion chromatography (chromatogram not shown). Inparallel, the flow through containing most of the active Am enzyme wascollected, concentrated, dialysed against 20 mM Na-phosphate, pH 7.0,and applied to a DEAE-Sepharose™ FF 16/10. Under the chosen conditions,active enzyme did not bind to the column and appeared in the flowthrough that was collected, concentrated and subjected to size exclusionchromatography. Even though AmCUT did not bind to DEAE-Sepharose™, mostof the contaminating proteins did and thus this step resulted in analmost pure enzyme. Active almost pure AmCUT was thus only found infraction X1 (flow through). All bound factions did not contain enzymeactivity.

For Am-Kr separated on Capto™-S, in contrast to AmCUT, Am-Kr did bind tothe cation exchanger in much larger quantity, although active enzyme wasalso found in the flow through. Fractions B3-B9 were active and werepooled (chromatograph not shown), concentrated and subjected to sizeexclusion chromatography as final purification step.

For CfCUT separated on Capto™-S, CfCUT did not bind to the cationexchanger at all. Thus, a similar purification approach was chosen asfor AmCUT cutinase. The active flow-through of CfCUT was collected,concentrated and diafiltrated against 20 mM sodium phosphate pH 7.0, andthe enzyme was loaded to a DEAE-Sepharose™ FF column (16/10). The flowthrough contained the active enzyme and was collected, concentrated andsubjected to size exclusion chromatography on Superdex™ 75 pg. ActiveCfCUT was exclusively found in fraction X1 (chromatograph not shown).None of the bound fractions contained active enzyme.

For Cf-TP separated on Capto™-S, in contrast to CfCUT, parts of Cf-TPdid bind to the cation exchanger, although active enzyme was also foundin the flow through. Fractions B3-B7 (chromatograph not shown) wereactive and were pooled, concentrated and subjected to size exclusionchromatography as final purification step.

In summary, using different techniques for enzyme purification, pureenzyme preparations were obtained and tested for their activity towardsa standard esterase substrate, pNP-palmitate. Enzymatic activities ofthe purified variants are summarized below:

-   -   KrCUT 197±3 U/mg    -   AmCUT 244±18 U/mg    -   Am-Kr 174±11 U/mg    -   CfCUT 26±1 U/mg    -   Cf-TP 30±2 U/mg

The active fraction of KrCUT (Superdex™ 75) was shown to have a nativemolar mass of about 28 kD (deducted from its retention time, using astandard curve), showing a single band in SDS-PAGE at about 27 kD (FIG.13A), which corresponds well with the theoretical molar mass for fulllength Kr (26 kD, after YebF cleavage).

AmCUT and Am-Kr behaved unusual in size exclusion chromatography, whichmade determining their native molar masses impossible, as the retentiontime of both enzymes did not correspond to their predicted molar mass.Instead, both enzymes were retained on the column, suggesting someinteraction of the enzymes with the column matrix. SDS-PAGE of Am-Kr(FIG. 13B) revealed two distinct species with molar masses of about 40kD (which is in agreement with the predicted molar mass for YebF-Am-Kr)and about 22 kD which is slightly below the predicted molar mass of fulllength Am-Kr (YebF removed, 26 kDa).

Example 19 Circular Dichroism Spectroscopy and Estimation of MeltingTemperature (Tm)

The enzymes purified in Example 18 were characterized to determine theirmelting temperatures.

19.1 Methods

CD spectra were recorded using a Jasco™ J-815 spectrometer operatingwith the Spectra™ Manager software. Temperature was controlled using aJasco™ PFD-452S peltier unit. Previously purified protein solutions wereadjusted to 0.15 mg/mL (if quantities allowed for) and CD spectra wererecorded between 190 and 250 nm using a Quartz cuvette (ID=0.1 cm).Blanks containing buffer only were prepared and used as baseline.Temperature dependent protein unfolding was monitored at 222 nm withthermo profiles ranging from 20 up to 90° C. (2° C. min-1). Samples werekept for 5 min at the respective maximum temperature and proteinrefolding was monitored using the same conditions as above reversing thethermo profiles. Thermodynamic parameters (Tm, ΔH, ΔS, ΔG) for thefolding/unfolding process, if applicable, were calculated using theSpectra™ Manager software.

19.2 Results

Variable temperature measurement using CD spectroscopy (monitored at 222nm) shows the unfolding of KrCUT with increased temperature (FIG. 14A).The melting temperature (Tm) was estimated to be 50.7° C.±0.1. KrCUTalso showed refolding to some extent (FIG. 14A, upper curve, going fromhigh to low temperature) with a midpoint transition temperature of 42.7°C.±0.2.

The Tm of Am-Kr was estimated to be 55.0° C.±0.1 (FIG. 14B). In contrastto KrCUT, Am-Kr did not show any refolding.

The Tm of CfCUT was estimated to be 65.2° C.±0.1 (FIG. 14C). CfCUT didnot show any refolding.

The CD signal of Cf-TP was a slightly low, probably due to lower proteinconcentration. The Tm of Cf-TP was estimated to be 56.6° C.±0.1 (FIG.14D). Cf-TP showed some refolding, but to a much lower extend whencompared to KrCUT.

The CD signal of AmCUT was too weak to allow for the estimation of a Tmfor this enzyme.

All enzymes showed the typical spectrum of alpha-helix dominantsecondary structure, as expected being hydrolases.

Example 20 Enzyme Substrate Profiles

The enzymes purified in Example 18 were characterized to determine theirsubstrate profiles on common esterase substrates.

20.1 Methods: Esterase Assays

Esterase activity was determined by monitoring the formation ofpara-nitrophenol (pNP) at 410 nm from various pNP-esters using a BeckmanUV spectrophotometer (model DU 640). Substrates were completelydissolved in 2-propanol (8 mM) and were then added to 50 mM Tris/HClbuffer pH 8.0 to give a final substrate concentration of 0.8 mM. Thereaction (1 mL) was started by adding an appropriate amount of enzyme.One enzyme unit (U) was defined as the amount of enzyme that produced 1μmol of product (pNP) per min; specific activity was expressed as U/mgprotein. A molar extinction coefficient of 15 mM⁻¹ cm⁻¹ was used for thecalculation.

20.2 Results

TABLE 11 Substrate profiles of enzymes tested Substrates KrCUT AmCUTAm-Kr CfCUT Cf-TP pNP-acetate (C2)  2  8  9 ± 1  6  5 pNP-butyrate (C4)26 ± 2 20 ± 2 75 ± 8 93 ± 8 80 ± 3 pNP-caprylate (C8) 25 ± 3 76 ± 2 100*11 ± 2 33 ± 1 pNP-decanoate (C10) 25 ± 3 80 ± 2 72 ± 2 40 ± 5 66 ± 8pNP-myristate (C14) 100* 100* 46 ± 8 47 ± 8 40 ± 4 pNP-palmitate (C16)98 ± 6 78 ± 2 49 ± 6 100* 100* *Relative activities in % for therespective cutinase, highest activity towards a specific substrate setto 100%

As can be seen in Table 11, the highest enzymatic activity for KrCUT andAmCUT was achieved with pNP-myristate, whereas the CfCUT and Cf-TPvariants showed the highest activity towards pNP-palmitate. In contrast,Am-Kr seemed to prefer the lower chain substrates with highest activitytowards pNP-caprylate. pNP-acetate seems to be a relatively poorsubstrate for all variants. KrCUT, AmCUT and Am-Kr showed preference forthe longer carbon chain substrates, whereas the CfCUT and Cf-TPsubstrate profile seemed to be more inhomogeneous, with preference forboth pNP-butyrate and pNP-palmitate.

Example 21 Determination of Enzyme Optimal Temperatures andThermostability

The enzymes purified in Example 18 were characterized to determine theiroptimum temperatures.

21.1 Methods: Temperature Optimum

For estimation of the temperature optimum, buffer containingpNP-palmitate (see esterase assay in Example 20.1) was pre-incubated toreach the desired temperature, enzyme was then added, and the formationof pNP was assayed. For this experiment, Tris/HCl buffer was replacedwith sodium phosphate buffer (50 mM, pH 8.0) to avoid a pH shift at thedifferent temperatures.

21.2 Results: Temperature Optimum

TABLE 12 Temperature dependence of the different enzymes testedTemperature (° C.) KrCUT AmCUT Am-Kr CfCUT Cf-TP 20 83 ± 5  92 ± 11 50 ±2 69 ± 2 54 ± 5 25 84 ± 4 100* 61 ± 2 100* 84 ± 9 30 83 ± 4 67 ± 3 69 ±2 63 ± 7 95 ± 5 35 84 ± 4 27 ± 4 78 ± 1 45 ± 5 100* 40 89 ± 6 15 ± 2 87± 2 38 ± 8 89 ± 6 45 100* — 100*  1 72 ± 7 50 90 ± 4 —  81 ± 10 — 37 ± 655 38 ± 3 — 37 ± 5 —  9 ± 1 60 15 ± 5 —  9 ± 1 — *Relative activities in% for the respective cutinase, highest activity at a specifictemperature set to 100%

As shown in the results above, the multi-domain enzyme KrCUT showedenzymatic activity at a broader range of temperatures than single-domainAmCUT and fellow multi-domain CfCUT (despite CfCUT having an higherestimated melting temperature than KrCUT, see Example 19.2).Interestingly, fusing the T/P-rich linker domain and the polymer bindingdomains from KrCUT to the cutinase catalytic domain of AmCUT, resultedin a chimeric enzyme (Am-Kr) having higher enzymatic activity at broaderrange of temperatures than AmCUT. Furthermore, replacing only theT/P-rich linker domain of CfCUT with the corresponding domain from KrCUTwas sufficient to produce a chimeric enzyme (Cf-TP) having enzymaticactivity at broader range of temperatures than CfCUT.

21.3 Methods: Thermostability

Enzyme thermostability was studied over a temperature range of 50-95° C.using the purified enzyme preparations. Aliquots were incubated at thedesired temperature for 5, 10, 30, 60, and 120 minutes, chilled on ice,and assayed for remaining activity towards p-NP-palmitate (pNPP) at roomtemperature.

21.4 Results: Thermostability

TABLE 13 Thermostability of the cutinase variants is shown in thefollowing table and graphs. Time (min) 50° C. 55° C. 60° C. 65° C. 70°C. KrCUT* 5 61 ± 5 45 ± 4 34 ± 4 19 ± 2 — 10 31 ± 4 26 ± 4 12 ± 2 — — 3012 ± 2 4 — — — 60 6 — — — — 120 — — — — — AmCUT* 5 46 ± 6 21 ± 4 3 — —10 18 ± 3 10 ± 2 — — — 30 5 — — — — 60 — — — — — 120 — — — — — Am-Kr* 596 ± 3  82 ± 17 19 ± 2 12 ± 1 — 10 85 ± 8 64 ± 3 15 ± 2 — — 30  75 ± 1556 ± 8  5 ± 1 — — 60  79 ± 12 42 ± 8 — — — 120 80 ± 9 30 ± 3 — — —cfCUT* 5 109 ± 3  72 ± 7 70 ± 6 65 ± 5 40 ± 5 10 118 ± 3   75 ± 10 75 ±7 64 ± 4 37 ± 6 30 129 ± 9  77 ± 9  56 ± 10 55 ± 1 33 ± 3 60 120 ± 6  65 ± 10 40 ± 4 30 ± 5 26 ± 3 120 125 ± 20 58 ± 2 28 ± 4 25 ± 4 15 ± 1Cf-TP* 5 68 ± 8 42 ± 4 35 ± 3 31 ± 3 — 10 43 ± 3 — — — — 30 42 ± 3 31 ±1 30 ± 3 25 ± 3 — 60 — — 22 ± 4 15 ± 2 — 120 — 21 ± 3 15 ± 3  9 ± 2 —*Relative activities in % for the respective cutinase, activity set to100% at time 0.

The results above show that single-domain enzyme AmCUT seemed to be veryunstable at higher temperatures, whereas all the multi-domain enzymes(particularly the CfCUT) showed higher thermostability. The lettervariant was also shown to have the highest melting temperature, however,the optimal temperature does not seem to reflect the enzyme's higherthermostability.

Example 22 Enzymatic Activity in the Presence of Organic Solvents

The enzymes purified in Example 18 were characterized to determine theirstability and activity in presence of diverse organic solvents.

22.1 Methods

Enzyme activity in various organic solvents was studied over aconcentration range of 1-30% at room temperature. pNP-palmitate wascompletely dissolved in 2-propanol (8 mM) and then added to 50 mMTris/HCl buffer pH 8.0 containing up to 30% organic solvent to give afinal substrate concentration of 0.8 mM. The reaction was started byaddition of enzyme and the formation of pNP was assayed as describedabove in Example 20.1.

22.2 Results

TABLE 14 Enzymatic activity in the presence of organic solvents Solventaddition to Solvent Relative enzymatic activity (%) assay conc. (v/v)KrCUT AmCUT Am-Kr CfCUT Cf-TP Buffer only 100*  100* 100* 100* 100*Methanol 10% 91 ± 12 116 ± 4   87 ± 10 115 ± 11 121 ± 14 20% 8 16 ± 2 60 ± 13 62 ± 3 94 ± 1 2-Propanol 5% 55 ± 11 60 ± 2  70 ± 10 70 ± 3 93 ±1 10% 8  9 ± 2 23 ± 2 17 ± 8 92 ± 2 Dimethyl sulfoxide 10% 106 ± 4  142± 12 114 ± 2  105 ± 11 150 ± 14 (DMSO) 20% 14 ± 2  53 ± 2 90 ± 8 107 ±1  146 ± 23 30% 2  7 ± 2 60 ± 5  57 ± 15 129 ± 18 Dimethylformamide 5%55 ± 11 149 ± 7  120 ± 11 101 ± 17 134 ± 10 (DMF) Acetone 1% 71 ± 2  170± 2  118 ± 1  92 ± 9 103 ± 12 5% 47 ± 7  126 ± 5   90 ± 14 95 ± 1 112 ±10 10% — 26 ± 7 74 ± 7 52 ± 5 109 ± 6  *Relative activities in % for therespective cutinase, activity with buffer only as negative control setto 100%

The results above show that the chimeric enzymes Am-Kr and Cf-TPimpressively exhibited increased tolerance to higher concentrations ofmost of the organic solvents tested. More particularly, the chimericAm-Kr enzymes exhibited higher relative activity in the presence ofmethanol (20% v/v), 2-propanol (10% v/v), dimethyl sulfoxide (DMSO; 20%or 30% v/v), and acetone (10% v/v), as compared to its correspondingwild-type enzymes (KrCUT and AmCUT). Similarly, the chimeric Cf-TPenzyme exhibited higher relative activity in the presence of all theorganic solvents tested, as compared to its corresponding wild-typeenzymes (KrCUT and CfCUT).

Example 23 pH Dependence of Enzymes

The enzymes purified in Example 18 were characterized to determine theirstability and activity at different pHs.

23.1 Methods: pH Optimum

The influence of different pH values (from 5.5 to 11) on the activitiesof the purified enzymes was examined in three buffer systems (sodiumacetate, sodium phosphate, and Tris-HCl) at room temperature usingpNP-palmitate (see esterase assay in Example 20.1) as a substrate at afinal substrate concentration of 0.8 mM.

23.2 Results

TABLE 15 pH dependence of each of the enzymes Relative activity (%) pHKrCUT AmCUT Am-Kr CfCUT Cf-TP 6.5 22 ± 1 35 ± 6 23 ± 2 13 ± 2 12 ± 1 7.053 ± 2 56 ± 6 46 ± 3 40 ± 4 22 ± 1 7.5 72 ± 4 56 ± 2 54 ± 3 65 ± 5 36 ±1 8.0 87 ± 6 38 ± 8 84 ± 2 62 ± 2 65 ± 7 8.5 100* 69 ± 4 88 ± 9 85 ± 688 ± 8 9.0 96 ± 4 94 ± 4 91 ± 5 93 ± 9 95 ± 3 9.5 76 ± 7 100* 100* 100*100* 10 37 ± 5 73 ± 2 47 ± 4 31 ± 1 35 ± 4 10.5 — 60 ± 2 10 ± 1 — —*Relative activities in % for the respective cutinase, highest activityat a specific pH set to 100%, sodium phosphate buffer (6.5-7.5),Tris/HCl (8.0-10.5)

Example 24 Stability of Enzymes Towards Oxidizing Agent

The enzymes purified in Example 18 were characterized to determine theirstability and activity after being exposed to an oxidizing agent (i.e.,hydrogen peroxide).

24.1 Methods: Stability in the Presence of Hydrogen Peroxide

Purified cutinases were incubated at room temperature in the presence of0, 30, 100, 300, 1500, and 3000 ppm of hydrogen peroxide. Residualenzyme activity was assayed (see Example 20.1) after 1, 2, 3, and 7 daysof incubation.

24.1 Results

TABLE 16 Enzyme stability following exposure to hydrogen peroxideRelative activity (%) H₂O₂ (ppm) Day 0 Day 1 Day 2 Day 3 Day 7 KrCUT* 30100 ± 9  90 ± 6 75 ± 3 75 ± 8 76 ± 2 100 104 ± 6  91 ± 6 88 ± 6 76 ± 266 ± 2 300 102 ± 4  87 ± 2 74 ± 1 63 ± 2 49 ± 4 1,500 102 ± 10 80 ± 4 67± 4 44 ± 2 11 ± 1 3,000 98 ± 9 60 ± 2 33 ± 1 20 ± 4 — AmCUT* 30 94 ± 6 —— — — 100 94 ± 6 — — — — 300  85 ± 12 — — — — 1,500 65 ± 6 — — — — 3,000 44 ± 10 — — — — Am-Kr* 30 92 ± 3  90 ± 15 82 ± 8 71 ± 4 63 ± 5 100 101± 3  97 ± 5 83 ± 2 73 ± 5 69 ± 4 300 100 ± 9  88 ± 5 83 ± 2  72 ± 10 61± 4 1,500 91 ± 8 97 ± 9  75 ± 15 73 ± 3 60 ± 6 3,000 86 ± 6 68 ± 9 63 ±4 67 ± 2 55 ± 3 CfCUT* 30 101 ± 22 89 ± 8 82 ± 8 76 ± 6 72 ± 7 100 92 ±5 114 ± 15 118 ± 15  92 ± 11 94 ± 7 300  93 ± 12 106 ± 20 118 ± 12 102 ±14 106 ± 13 1,500 126 ± 1  106 ± 12 109 ± 2  95 ± 4  54 ± 16 3,000 128 ±18 115 ± 2   99 ± 23 60 ± 6 35 ± 1 Cf-TP* 30 97 ± 1 89 ± 4 78 ± 3 86 ± 265 ± 7 100 98 ± 6 91 ± 3 89 ± 7 94 ± 7 74 ± 6 300 95 ± 6 88 ± 3  85 ± 1298 ± 3 77 ± 5 1,500 95 ± 9 89 ± 3 88 ± 6 87 ± 4 68 ± 7 3,000 80 ± 2 74 ±5 77 ± 2 78 ± 5 57 ± 5 *Relative activities in % for the respectivecutinase, activity with buffer only as negative control set to 100% atday 0.

As shown above, all of the multi-domain enzymes that were tested (i.e.,KrCUT, Kr-Am, CfCUT, and Cf-TP) retained some level of activity after 1or more days exposure to hydrogen peroxide, whereas the single-domainenzyme AmCUT did not. Furthermore, chimeric enzymes (Kr-Am and Cf-TP)exhibited increased resistance to hydrogen peroxide, as compared totheir corresponding wild-type multi-domain enzymes (KrCUT and CfCUT).For example, after exposure to 3000 ppm hydrogen peroxide for 7 days, noactivity was detected for the wild-type KrCUT enzyme. In contrast, thechimeric enzyme Am-Kr (containing the cutinase catalytic domain of AmCUTon the backbone of KrCUT), retained 55% of its activity under the sameconditions. Moreover, the relative activity of CfCUT was reduced from128% at day 0 to 35% at day 7 of exposure to 3000 ppm hydrogen peroxide.In contrast, the relative activity of the chimeric enzyme Cf-TP(containing the T/P-rich linker domain of KrCUT on the backbone ofCfCUT) went from 80% to 57% under the same conditions.

Example 25 Summary of Biochemical Properties of the Enzymes

A summary of some of the results shown in Examples 19-24 is providedbelow.

TABLE 17 Summary of biochemical properties of the tested enzymes Mol.Mol. Temp. Mass Mass opt. Spec. Act. Tm Enzyme (native)¹ (SDS) (° C.) pHopt. (U/mg)² (° C.)³ KrCUT 28,000 27,000 45 8.5 197 ± 3  51 ± 1 AmCUTn.a.⁴ 20,000 25 9.5 244 ± 18 n.d.⁵ Am-Kr n.a.⁵ 22,000 45 9.5 174 ± 11 55± 1 CfCUT⁶ 53,000 27,000 25 9.5 26 ± 1 65 ± 1 Cf-TP⁷ 42,000 25,000 359.5 30 ± 2 57 ± 1 ¹Estimated by retention time of the active fraction onSuperdex ™ 75 pg using a standard curve ²Towards pNP-palmitate (standardassay) ³Melting temperature of the enzyme, estimated usingCD-spectroscopy ⁴Not applicable, Am and Am-Kr showed some interactionwith the resin and thus a delayed retention time ⁵Not determined, Amconcentration was too low to give a good signal in CD spectroscopy ⁶Cfshows a higher molecular mass native than in SDS-PAGE, which may be dueto dimer formation ⁷Cf-TP showed more than one band in SDS-PAGE: 25,000and 28,000 Da.

Example 26 Assessment of Ochratoxin a Degradation Ability by the Enzymes

The enzymes purified in Example 18 were tested for their ability todegrade the mycotoxin Ochratoxin A. In general, the mycotoxin wasincubated with different concentrations of the enzyme, and the remainingmycotoxin concentration, as well the respective degradation products,were monitored by LC-MS over time (where possible/applicable).

26.1 Methods: Mycotoxin Testing

Reactions were performed in 1 mL volumes in glass HPLC vials comprisingthe mycotoxin to be analyzed and the enzyme to be tested. For OchratoxinA (Petromyces albertensis, 01877 Sigma), 50 μL of a 100 ppm stocksolution in methanol (final concentration of 5 ppm) was added to 910 μLof distilled water and 40 μL of the enzyme to be tested. In controlreactions, the volume of enzyme was substituted with distilled water.The reactions were incubated in an Eppendorf thermomixer at 37° C. withshaking at 750 rpm. 100 μL samples were withdrawn at 1, 4 and 24 h forchromatographic analysis.

Mycotoxin analysis was carried out using a Shimadzu LCMS-2020 equippedwith a UV/vis detector. Chromatographic separation of 10 μL injectedsample was carried out at 30° C. on a Kinetex 2.6 μm XB-C18 column 100 Å(50×2.1 mm) using a binary gradient. For Ochratoxin A, eluent Aconsisted of water and eluent B consisted of acetonitrile and the UVdetection wavelength was 360 nm. Gradient elution at a flow rate of 0.4mL/min was performed as follows: 0-2 min, 10% B; 2.01-10.0 min, 10 to80% B; 10.01-12.0 min, 80% B; 12.01-15 min, 10% B. The electrosprayinterface (ESI) was operated simultaneously in positive and negativemode, scanning from m/z 50-1,000.

26.2 Results

The degradation of Ochratoxin A was monitored by HPLC-UV at 360 nm andLCMS. Possible degradation products that would arise from eitherhydrolysis of the amide bond or hydrolysis of the lactone moiety areshown below.

Chemical Structures of Ochratoxin a and Hypothetical DegradationProducts

The five enzymes tested were screened for their ability to degrade 5 ppm(5,000 ppb) Ochratoxin A at 37° C. The results are summarized in Table18.

TABLE 18 Results of Enzyme testing for Ochratoxin A degradation Residualochratoxin A Ochratoxin A (%)² Degradation³ per Enzyme Activity (U)¹ 1 h4 h 24 h U and h (μmol) KrCUT 3.272 63 17 7 0.02 Am-Kr 0.072 6 0 0 3AmCUT 0.048 59 20 6 2 CfCUT 0.056 11 2 0 3 Cf-TP 0.002 5 0 0 98¹Activity used in assay towards p-nitrophenyl palmitate (standard assay)²Relative to Ochratoxin A incubated without enzyme ³Describes thepotency of 1 U of enzyme (pNP standard test) to degrade a specificamount of ochratoxin per hour of incubation at 37° C.

Degradation of Ochratoxin A was observed with all enzymes, even after 1h incubation. The chimeric enzymes AM-Kr and Cf-TP degraded 94-95% ofthe Ochratoxin A after 1 hours, and 100% after 4 hours. Cf-TP showed thehighest “potency” in terms of the amount of Ochratoxin A that 1 U theenzyme (pNP standard test) can degrade per hour of incubation at 37° C.

Example 27 Alignment and Phylogenic Analyses of Multi-Domain CutinaseEnzymes

A database search for other multi-domain enzymes using KrCUT (SEQ ID NO:6) as query sequence revealed five hits, as shown in Table 19.

TABLE 19 Native cutinase orthologs having multi-domain organizationEnzyme Query name Organism coverage Identity Accession SEQ ID NO:ssKrCUT Kineococcus radiotolerans 100% 100% ABS05574.1 1 SRS30216 = ATCCBAA-149 ssCbCUT2 Cellulomonas bogoriensis 95% 60% KGM14008.1 25 69B4 =DSM 16987 ssCcCUT Cellulomonas cellasea DSM 98% 56% KGM03336.1 15 20118ssCbCUT1 Cellulomonas bogoriensis 99% 55% KGM14009.1 14 69B4 = DSM 16987ssCfCUT Cellulomonas flavigena DSM 99% 56% ADG75999.1 13 20109

A multiple sequence alignment was performed for the enzymes listed inTable 19, after omitting their predicted N-terminal signal sequences, asshown in Table 20. The results of the alignment are shown in FIG. 15A,and a phylogenic tree is shown in FIG. 15B.

TABLE 20 KrCUT orthologs without their predicted N-terminal signal sequences Amino acid sequence(cutinase catalytic domains are in bold; Enzymelinkers are highlighted in black; SEQ namepolymer binding domains are underlined) ID NO: KrCUTATCSDVDVVFARGTGETPGLGVVGGPFVRSLTGELSDRTVTSHA  6VDYAASSSQASAGPGATAMSAHVREVAAACPSTRFVLGGYSQGATVTDIALGIRTGTTTGTPVPAELAGRVAAVVVFGNPLGLSGRTIATASSTYGPKSKDYCNSSDSVCGSAPKTGTGGHLSYASNGSTTD

AVSLYGRAYARGSRDSLGATSSYNVVSLQQVEGGWRLVTAC CbCUT2AGSCPDVQVVFARGTGERAGLGIIGRPFARALADELPGMTVTSH 46AVDYAAAASQRSAGPGATAMTDHVTAMAARCPGTQFVLGGYSQGATVTSIALGIRAGTTTGRAIPDELSDRVAAVVVFGNPLGMRGQTIASASRTYADRAKDYCNSGDSICGRQPSTGRGTHTGYATNGSTT

VSEHVEARRAIRGLTRAYARGTLEDIGRLRSTEEVSLRRSGTFS WTPTASC CcCUTAPACADVEVLFARGTGEAPGLGVLGTPFVRSVTSALSDRTVTSY 47AVNYAAESSQRSAGPGATDLTNHLTATAAACPGTRFVLGGYSQGATVVDLALGIRTGTTTGTAIPAALEPRVAAIVVFGNPLGISGRTIATASPTYAARARDFCATGDPVCGGGSSFAAHLAYRTNGDVTAG ADFAAGLARA

GAACVRASTRAHVEAGRAERRHGIAYATGSGDRIGWV SSFVRVSVQQTADGWERVLSC CbCUT1TGDCPDVHVVFARGTGEPRGLGIVGRPFVSDLGDALPTMTVTSY 48AVNYSANASQTSAGPGAGDMTSHVTSMAARCPGTQFVLGGYSQGATVTSIAVGARSTSIRSRVLPANLEPRVAAVVVFGNPLGLTRRTIASEAPAYAAKSRDYCNRSDTVCGGRGDARGGHLAYVSNGSVAD

SRDPLGRLTSSDLVSLQRTGQDSWSVVPAC CfCUTAPACPDVELVFARGTGEAAGLGIVGRPLERALAAELPGRTVVAT 28AVDYAASSSQASAGPGSGDMVAKVRSRAAACPGTQFVLGGYSQGATVTDLALGIRTGVTAGTALPEDLAARVAAVVVYGNPLGLTRRTIAQAAPAFATRTVEYCNAGDPVCEPGGGRFTAHITYATNGTVLE

SLQHVRDGRAYPLWMRTYARGSGDPLGVLSSRTVVSLQADGTDT WRKVAAC

The threonine/proline contents of the T/P-rich linker domains of four ofthe enzymes listed above are shown in Table 21.

TABLE 21 Threonine/proline content of selected multi-domain cutinases #of Enzyme Amino acid sequence SEQ ID T or P Linker T/P nameof T/P-rich linker NO: residues length content KrCUT TPTTPTPTPTPTPVPTT 3 16 17 94.1% CbCUT2 NPPEAAPPTAPPTTPPTTPAPP 37 16 22 72.7% CcCUTGLARATTVPVPTPTVPGTPTPSP 40 17 28 60.7% TAPGTPTPVPTTSPTPAPSPTAP GAA CfCUTPAPTPTPGPTGTPAPVPSAEPTP 31 29 49 59% APGDT

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

The scope of the claims should not be limited by the preferredembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

REFERENCES

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What is claimed is:
 1. An isolated chimeric recombinant protein havingcutinase activity, said protein comprising a cutinase catalytic domainand a polymer binding domain operably linked by a proline/threonine-richlinker domain, wherein the proline/threonine-rich linker domaincomprises at least 50% proline or threonine residues over a stretch of15 to 55 consecutive amino acids.
 2. The isolated chimeric recombinantprotein of claim 1, wherein the proline/threonine-rich linker domaincomprises an amino acid sequence at least 80% identical to SEQ ID NO: 3,31, 37, or
 40. 3. The isolated chimeric recombinant protein of claim 1,wherein at least two of the cutinase catalytic domain, the polymerbinding domain, and the proline/threonine-rich linker domain originatefrom native enzymes of different species.
 4. The isolated chimericrecombinant protein of claim 3, wherein the polymer binding domain andthe proline/threonine-rich linker domain are selected from amulti-domain cutinase from the species Kineococcus radiotolerans,Cellulomonas flavigena, Cellulomonas bogoriensis, and Cellulomonascellasea.
 5. The isolated chimeric recombinant protein of claim 3,wherein the polymer binding domain is from a poly(3-hydroxybutyrate)(PHB) depolymerase of the species Paucimonas lemoignei, Thermobifida sp.BCC23166, Comamonas testosterone, Thermobifida fusca, or Nocardiopsisdassonvillei.
 6. The isolated chimeric recombinant protein of claim 1,which is recombinantly produced fused to a heterologous carrier proteinor heterologous tag that enables secretion of said recombinant protein,and/or facilitates purification or detection of said recombinantprotein.
 7. The isolated chimeric recombinant protein of claim 6,wherein said carrier protein is YebF.
 8. A composition comprising: (i)an isolated recombinant protein having cutinase activity, said proteincomprising a cutinase catalytic domain and a polymer binding domainoperably linked by a proline/threonine-rich linker domain, wherein theproline/threonine-rich linker domain comprises at least 50% proline orthreonine residues over a stretch of 15 to 55 consecutive amino acids;and (ii) a bleaching agent and/or an organic solvent, wherein saidrecombinant protein retains said cutinase activity when present in saidcomposition, and wherein the composition is: a cleaning product, adegreaser, a disinfectant, a bleaching product, a product forcontrolling biofilm, or any combination thereof; feed or a feedadditive; a product for inactivating mycotoxin; a product forbioscouring or other treatment of fabrics or textiles; a product forcatalyzing trans-esterification reactions; a product for vegetable oilextraction; or a product for enhancing oil recovery.
 9. The compositionof claim 8 comprising a bleaching agent which is: hydrogen peroxide, aperoxide other than hydrogen peroxide, a non-peroxide oxidizing agent,ozone, sodium percarbonate, sodium perborate, and any combinationthereof.
 10. The composition of claim 8 comprising a peroxide.
 11. Thecomposition of claim 8 comprising a bleaching agent which is: sodiumdithionite, sodium dithionite, sulfur dioxide, a sulfite, a bisulfite, asodium borohydride, or any combination thereof.
 12. The composition ofclaim 8 comprising an organic solvent which is: methanol, 2-propanol, oran alcohol other than methanol or 2-propanol, dimethyl sulfoxide (DMSO),dimethylformamide (DMF), or acetone.
 13. The composition of claim 8having a pH between 7.5 and 9.5.
 14. The composition of claim 8, furthercomprising: a detergent, a surfactant, a buffer, a chelator, or anycombination thereof.
 15. The composition of claim 8, further comprisinga chelator which is EDTA or GLDA.
 16. The composition of claim 8,further comprising a polyol and/or boric acid as a stabilizer.
 17. Thecomposition of claim 8, further comprising an additional recombinantenzyme which is: a lipase, an esterase, a pectate lyase, a pectinase, acutinase, a cellulase, a hemicellulose, an amylase, or any combinationthereof.
 18. The composition of claim 8, wherein theproline/threonine-rich linker domain comprises an amino acid sequence atleast 80% identical to SEQ ID NO: 3, 31, 37, or
 40. 19. The compositionof claim 8, wherein at least two of the cutinase catalytic domain, thepolymer binding domain, and the proline/threonine-rich linker domainoriginate from native enzymes of different species.
 20. The compositionof claim 8, wherein: (a) the proline/threonine-rich linker domain isselected from a multi-domain cutinase from the species Kineococcusradiotolerans, Cellulomonas flavigena, Cellulomonas bogoriensis, andCellulomonas cellasea; or (b) the polymer binding domain is selectedfrom a multi-domain cutinase from the species Kineococcus radiotolerans,Cellulomonas flavigena, Cellulomonas bogoriensis, and Cellulomonascellasea; or from a poly(3-hydroxybutyrate) (PHB) depolymerase of thespecies Paucimonas lemoignei, Thermobifida sp. BCC23166, Comamonastestosterone, Thermobifida fusca, and Nocardiopsis dassonvillei.